An ABC Transporter Mutation Alters Root Exudation of Phytochemicals That Provoke an Overhaul of Natural Soil Microbiota

Abstract

Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.

Full text

An ABC Transporter Mutation Alters Root Exudation
of Phytochemicals That Provoke an Overhaul of Natural
Soil Microbiota1[C][W][OA]
Dayakar V. Badri2,NairaQuintana
2, Elie G. El Kassis, Hye Kyong Kim, Young Hae Choi, Akifumi Sugiyama,
Robert Verpoorte, Enrico Martinoia, Daniel K. Manter, and Jorge M. Vivanco*
Center for Rhizosphere Biology and Department of Horticulture and Landscape Architecture (D.V.B., E.G.E.K.,
A.S., J.M.V.), and Department of Chemistry (N.Q.), Colorado State University, Fort Collins, Colorado 80523;
Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, 2300 Leiden, The
Netherlands (H.K.K., Y.H.C., R.V.); Zurich-Basel Plant Science Center, Institute of Plant Biology, Molecular Plant
Physiology, University of Zurich, CH–8008 Zurich, Switzerland (E.M.); and United States Department of
Agriculture-Agricultural Research Service, Soil-Plant-Nutrient Research Unit, Fort Collins, Colorado 80526
(D.K.M.)
Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-
binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven
Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations,
only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared
with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the
mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased
phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots
revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some
sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with
Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial
community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria
and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report
how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing
that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community
of organisms as well.
The diversity of the microbial (bacterial and fungal)
communities in soil is extraordinary; 1 g of soil con-
tains more than 10 billion microorganisms belonging
to thousands of different species (Rosello
´-Mora and
Amann, 2001). Soil microbial populations are involved
in a framework of interactions known to affect key
environmental processes like biogeochemical cycling
of nutrients, plant health, and soil quality (Pace, 1997;
Barea et al., 2004; Giri et al., 2005). Most of the dynamic
soil microbial interactions happen near the plant roots
and root soil interface, an area called the rhizosphere
(Lynch, 1990; Barea et al., 2002; Bais et al., 2006;
Prithiviraj et al., 2007). Rhizosphere microbial com-
munities differ between plant species (Priha et al.,
1999; Innes et al., 2004; Batten et al., 2006), between
ecotypes/chemotypes within species (Kowalchuk
et al., 2006; Micallef et al., 2009), between different
developmental stages of a given plant (Mougel et al.,
2006; Weisskopf et al., 2006), and from those present in
bulk soil (Broz et al., 2007). Different root types can
also cultivate specific microbes (Lilijeroth et al., 1991;
Yang and Crowley, 2000; Baudoin et al., 2002), a
response that has generally been attributed to the
microenvironments surrounding a root and the vary-
ing ability of specific root types to uptake nutrients
from soils and secrete exudates. Recent evidence
1
This research was supported by grants from the National
Science Foundation to J.M.V. (MCB–0542642). The contribution of
E.M. was supported by the Swiss National Foundation within the
NCCR “Plant Survival” and the European Union project Plant
Transporters (HPRN–CT–2002–00269). A.S. is a recipient of a Japan
Society for the Promotion of Science abroad fellowship.
2
These authors contributed equally to the article.
* Corresponding author; e-mail j.vivanco@colostate.edu.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jorge M. Vivanco (j.vivanco@colostate.edu).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a sub-
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.147462
2006 Plant PhysiologyÒ,December 2009, Vol. 151, pp. 2006–2017, www.plantphysiol.org Ó2009 American Society of Plant Biologists

suggests that specific plant species support a highly
coevolved soil fungal community, and this process is
mediated by root-secreted compounds (Broeckling
et al., 2008). Rhizosphere interactions are initiated by
the release of compounds from different organisms,
and it is believed that carbon compounds secreted by
roots act as substrates for certain species of microbes in
the rhizospshere (Morgan et al., 2005).
Root exudates are released into the rhizosphere by
three major pathways: diffusion, ion channel, and
vesicle transport (Bertin et al., 2003). Recent evidence
has implicated ATP-binding cassette (ABC) trans-
porters in the secretion of phytochemicals present in
the root exudates of Arabidopsis (Arabidopsis thaliana)
and other plants (Loyola-Vargas et al., 2007; Sugiyama
et al., 2007; Badri et al., 2008; Badri and Vivanco, 2009).
ABC transporters are the largest family of membrane
transport proteins found in all organisms from bacte-
ria to humans (Higgins, 1992). These transmembrane
proteins use the energy of ATP to pump a wide variety
of substrates across the membranes, including pep-
tides, carbohydrates, lipids, heavy metal chelates, in-
organic acids, steroids, and xenobiotics (Goossens
et al., 2003). ABC transporters are also involved in
plant disease resistance at the leaf level (Kobae et al.,
2006; Stein et al., 2006).
There is accumulating evidence that root exudates
play a role in establishing specific interactions with
particular microbes in the rhizosphere (legume’s sym-
biotic interaction with rhizobia, interaction of plants
with mycorrhizae, and plant-growth-promoting rhi-
zobacteria [PGPR]; Nagahashi and Douds, 2000; Bais
et al., 2006, 2008; Prithiviraj et al., 2007; Rudrappa
et al., 2008). However, how root exudation processes
that result in large-scale changes to the surrounding
soil microbial community compared to individual
microbes have not been determined, although some
recent reviews have referred to it as a biological
frontier (O’Connell et al., 1996; Kuiper et al., 2004;
Ryan et al., 2009). In contrast, gene deletions and
overexpression of specific genes in plants have been
shown to attract or deter specific microbes (Widmer,
2007), herbivores, or their predators (Baldwin et al.,
2006; Pandey and Baldwin, 2007; Mitra and Baldwin,
2008), and recently it has been shown that mutations in
nonpigment floral chemistry genes affect flower visi-
tation by native pollinators (Kessler et al., 2008). Thus,
it is possible that gene expression manipulation lead-
ing to an altered spectrum of root exudates can influ-
ence the widespread community of soil organisms
surrounding a plant. Using all available information
described above, we present the most comprehensive
study on the effect of a single gene mutation in an ABC
transporter involved in root secretion of phytochem-
icals by Arabidopsis on the natural and coevolved soil
microbial composition. We further determine the com-
pounds that are likely to have an effect on moderating
the microbial composition and characterized specific
and natural microbes that interact with Arabidopsis in
the soil by employing pyrosequencing technology.
RESULTS
Microbial Diversity Analyses
We analyzed the soil microbial community structure
supported by Arabidopsis wild type (Columbia-0
[Col-0]) and seven ABC transporter mutants (abca7,
abcc2,abcg30,abcg34,abcg35,abcb1, and abcb4; Verrier
et al., 2008) grown in Arabidopsis-accustomed soil for
two subsequent generations. At the onset of this
experiment (generation 0), significant differences in
the soil microbial community structure were observed
(Fig. 1; Supplemental Tables S1 and S2), presumably
associated with natural spatial heterogeneity in the
soil, despite our attempts to homogenize the starting
soil. However, after one generation, this heterogeneity
disappeared and there were no significant differences
in either bacterial or fungal community structure
between any of the treatments (Fig. 1; Supplemental
Tables S1 and S2). Following the second generation, we
found that the ABC transporter mutant abcg30 signif-
icantly affected both fungal and bacterial microbial
community structure compared with other ABC trans-
porter mutants and the wild type based on an multi-
response permutation procedure (MRPP) analysis of
the entire automated ribosomal intergenic spacer anal-
ysis (ARISA) profile (Fig. 1; Supplemental Table S1).
Based on the ARISA analysis, eight fungal opera-
tional taxonomic units (OTUs) decreased significantly
in the soil when abcg30 was grown, compared to the
wild type and the negative control (no plant). Simi-
larly, 14 bacterial OTUs decreased and four bacterial
OTUs increased when abcg30 was grown (Supplemen-
tal Fig. S1). Among the eight fungal OTUs that de-
creased significantly, four of them also decreased in
the negative control (no plant; Supplemental Fig. S1A).
This result indicates that the other four OTUs (H72,
H117, H123, and H129) decreased specifically due to
the absence of abcg30. Similarly, among the 14 bacterial
OTUs that decreased significantly with abcg30, three
OTUs (H116, H170, and H241) increased in the nega-
tive control compared with the wild type (Supple-
mental Fig. S1B). These results suggest that the three
OTUs increased in the negative control due to envi-
ronmental factors present in the greenhouse. Two
other OTUs (H155 and H255) decreased in both
abcg30 and the negative control compared to the wild
type. In addition, four OTUs (H90, H19, H141, and
H164) increased significantly in abcg30 compared with
the wild type, but one OTU (H90) also increased in the
negative control. These results suggest that the root
exudates of the wild type and the ABC transporter
mutant abcg30 might have a different composition of
phytochemicals, which impacted the microbial com-
munity composition of the native soils.
Chemical Analysis of Root Exudates
The root exudates of Arabidopsis wild type and all
ABC transporter mutants were analyzed in this study
via NMR spectroscopy coupled with multivariate data
ABC Transporter Mutant Alters Natural Soil Microbiota
Plant Physiol. Vol. 151, 2009 2007

analyses. In total, based on 1H-NMR and other spectral
analyses, we identified 33 compounds in the wild-type
root exudates, including organic acids, amino acids,
sugars, flavonols, phenolics, anthocyanidins, and in-
dole compounds (Supplemental Figs. S2–S10). Princi-
pal component analysis (PCA) of the root exudates
using the J-resolved NMR signals showed that the 10
Arabidopsis lines used in this study fell into seven
statistically different groups (Fig. 2A). The root exu-
dates of abcg30 appeared to be unique (Fig. 2A) and
showed the lowest similarity to the wild type (Fig. 2B).
Compared to the wild type, the following compounds
were present in higher concentrations in the root
exudates of abcg30: benzoic acid, salicylic acid, syrin-
gic acid, tartaric acid, lactic acid, a-linolenic acid,
cyanidin, sinapoyl malate, Val, and indole 3-acetic
acid. In contrast, lower amounts of some sugars (raf-
finose, Glc, Fru, and mannitol) were present (Supple-
mental Table S3).
Analyzing the Pleiotropic Effect of abcg30 Mutation at
the Genome Level
Based on the comparison of the root exudates pro-
files of all ABC transporter mutants, we found that
abcg30 had increased phenolics and fewer sugars
compared to the wild type. We hypothesized that the
observed differences (phenolics versus sugars) in
abcg30 root exudates might not be under the direct
control of the ABCG30 transporter but are probably
the pleiotropic effects of the mutation. To elucidate the
pleiotropic effects of abcg30 mutation, we performed
whole-genome expression analyses on abcg30 roots
compared with wild-type roots using the Affymetrix
GeneChip Arabidopsis ATH1 genome array chip. Out
of 22,810 genes on the chip, 355 (1.5%) genes were up-
regulated .1.5-fold, and 156 (0.7%) genes were down-
regulated to ,0.5-fold in abcg30 compared to the wild
type. We focused our analyses on selected genes
involved in transport, secondary metabolism bio-
synthesis, and transcription factors (Supplemental
Table S4). We observed that 10 genes involved in trans-
port, including lipid transporters (At4g22460 and
At3g22120) and ABC transporters (At5g44110 and
At2g26910), and 16 genes involved in secondary
metabolism, including phenylproanoid (At2g23910,
At1g65060, and At1g67980) and flavonoid biosnthesis
genes (At3g55120, At5g08640, At5g05270, At5g07990,
and At5g13930) were up-regulated significantly in
abcg30 compared to the wild type. Similarly, six genes
involved in transport, including sugar transporters
(At1g08920 and At4g04760) and a mannitol trans-
porter (At4g36670), and nine genes involved in sec-
ondary metabolism, such as terpene biosynthesis
(At4g20230, At3g31415, and At5g42600), were down-
regulated in abcg30 compared to the wild type. In
addition to transporters and secondary metabolism
biosynthesis genes, we found that some transcription
factors and genes belonging to different functional
categories, such as primary metabolism, signal trans-
duction, cell growth, and cell division, defense re-
sponses and genes of unknown function were also
differentially (both up- and down-regulated) ex-
pressed in abcg30 compared with the wild type (Sup-
plemental Table S4 and Figure S11). The microarray
data were also deposited to a permanent public re-
pository, ArrayExpress, under the accession number
E-TABM-821.
In Vitro Analysis of the Effect of Arabidopsis Wild-Type
and abcg30 Exudates on Native Soil Microbes
Due to the differences in phytochemical composi-
tion in the root exudates, we examined whether wild-
Figure 1. NMS analysis of fungal and
bacterial community profiles of Arabi-
dopsis wild type and ABC transporter
mutant rhizospheric soil samples con-
ducted using the relative Sorenson
distance measure. All generation sam-
ples were analyzed simultaneously. A,
Fungi; B, bacteria. G0, zero generation
(before seeding); G1, first generation;
G2, second generation; Ler, Landsberg
erecta ecotype; T, negative control (soil
without plant).
Badri et al.
2008 Plant Physiol. Vol. 151, 2009

type and abcg30 root exudates affected the in vitro
growth of natural Arabidopsis soil microbes using
standard serial dilution techniques. The abcg30 exu-
dates significantly reduced both the number and
growth rate of the fast-growing, culturable soil mi-
crobes. For example, after 24 h, 2.8 3107colonies were
visible on Col-0 exudates amended plates, and no col-
onies were observed on abcg30 exudates amended plates,
whereas, at the termination of the experiment (48 h), only
7.1 3106colonies could be observed on the abcg30
amended plates, but Col-0 amended plates showed
complete bacterial lawns (Supplemental Table S5).
Taxa Identification Using Pyrosequencing
To further characterize the specific taxa associated
with abcg30 and wild-type root exudates, we charac-
terized the soil microbial communities after the 2nd
generation of plant growth by pyrosequencing of
rRNA libraries. A total of 160 fungal (Fig. 3A) and
2,489 bacterial (Fig. 3B) sequence reads were obtained
Figure 2. A, PCA of Arabidopsis wild type (Col-0) and ABC transporter
mutant root exudates using projected one-dimensional J-resolved
spectrum. B, Percentage of matched two-dimensional J-resolved signals
between wild type (Col-0) and Arabidopsis ABC transporter mutant root
exudates. a, Col-0; b, abca7;c,dtx12;d,abcc2;e,abcg30;f,abcg34;g,
abcg35;h,abcb1;i,abcb4;j,abcb27.
Figure 3. Fungal (A) and bacterial (B) OTUs present in the 2nd
generation of Atabcg30 and wild-type soils determined by rRNA
pyrosequencing. Green, abcg30; blue, wild type (Col-0); pink, shared.
C, Total estimated species richness (ACE and Chao) and Shannon
diversity index (H). D, Significance of microbial libraries community
profile comparison using E-libshuff. [See online article for color version
of this figure.]
ABC Transporter Mutant Alters Natural Soil Microbiota
Plant Physiol. Vol. 151, 2009 2009

from the wild-type and abcg30 soils. Although there
appeared to be no difference in either fungal or
bacterial total OTU richness or diversity (Shannon-
diversity index) between the two soils (Fig. 3C), each
library was characterized by a high number of unique
(i.e. below the detection level of 1 per 2,689 reads or a
relative abundance #0.037%) taxa resulting in statis-
tically different communities (Fig. 3D). For example, at
a genetic distance of 1%, 84 fungal and 1,661 bacterial
OTUs were unique to the abcg30 community, whereas
90 fungal and 1,879 bacterial OTUs were unique to the
wild-type community.
From this analysis, the relative abundance of 105
bacterial OTUs differed significantly (P,0.05) be-
tween the wild type and abcg30 (Fig. 4A, B). Unam-
biguous identification of the various OTUs to the
species/strain level is frequently not possible due to
either high homology of the rRNA sequences to mul-
tiple species or missing reference sequences in the
relevant databases. In this study, however, many of the
abcg30 up-regulated OTUs appear to be closely related
to known PGPRs or otherwise beneficial bacteria.
These include one (species) Microbacterium sp., one
Nocardioidaceae sp., one Pseudonocardia sp., one Flex-
ibacteraceae sp., three (species) Sphingobacteriales
spp., one Methylibium sp., two Duganella spp., one
Pseudoxanthomonas sp., one Flavobacterium sp., six
Brevundimonas spp., one Cystobacter sp., three Rhodo-
Figure 4. Relative abundance of bac-
terial OTUs (1% dissimilarity) signifi-
cantly up- (A) or down-regulated (B) in
the abcg30 mutant soils compared to
the wild type. Taxonomic assignments
were made with the RDP II naı
¨ve
Bayesian classifier (80% confidence
threshold) using a single representative
sequence for each OTU. Col-0, black
bars; abcg30, gray bars.
Badri et al.
2010 Plant Physiol. Vol. 151, 2009

bacteriaceae spp., two Bradyrhizobium spp., and one
Paracoccus sp. The Microbacterium, Nocardioidaceae,
Flexibacteraceae, Sphingobacteriales, and Flavobacte-
rium spp. are of special interest because they play a
role in heavy metal (Ni and S) remediation (Idris et al.,
2004), whereas the Pseudonocardia,Methylibium,Duga-
nella, and Pseudoxanthomonas spp. are involved in
detoxifying toxic compounds like ether pollutants,
dinitrotoluene, bioplastics, and methylated aromatic
compounds (Grech-Mora et al., 1996; Kaplan and
Kittis, 2004; Vainberg et al., 2006; Kane et al., 2007;
Kim et al., 2008). A variety of bacteria associated with
nitrogen fixation were also up-regulated in the abcg30
soil, including six Brevundimonas spp., two Bradyrhi-
zobium spp.. and one Paracoccus sp. In contrast, the
wild-type soil preferentially supported a much smaller
number of potential PGPBs or otherwise beneficial
bacteria. These include three Brevundimonas spp., one
Bradyrhizobium sp., one Lysobacter sp., and one Bacillus
sp., which are different OTUs than the ones cultivated
by abcg30 based on their genetic distance (.1%) and
phylogenetic analysis with their nearest known neigh-
bors (Supplemental Fig. S12). Differences were also
observed for the fungal community structure in the
soils surrounding the wild type and the abcg30 mutant
(Fig. 5). Four fungal OTUs were significantly up-
regulated (Ciliophora sp., one Basidiomycota sp., one
Scenedesmus sp., and one Trebouxiophyceae sp.), and
five were significantly down-regulated (two Ascomy-
cota spp., two Mortierella spp., and one Ciliophora sp.).
Additionally, we determined that one Xylella sp. was
enriched in the soil cultured by abcg30 compared to
wild-type-grown soil, and a similar observation was
found for Mycobacterium sp.
DISCUSSION
The impact of plant species and closely related
variants (i.e. ecotypes) on rhizobacterial communities
has been well documented (Dalmastri, 1999; Mazzola
et al., 2004; Micallef et al., 2009). In this study, we
showed that a single gene mutation (ABC transporter,
abcg30) involved in root exudation influences the soil
microbial community. We analyzed seven Arabidop-
sis ABC transporter mutants, whose putative trans-
porters were highly expressed in root cells (Badri
et al., 2008), for differences in their root exudates and
their ability to influence the microbial community in
Arabidopsis-accustomed soil. Previously, it has been
shown that Arabidopsis fails to support the fungal
community of soils to which it is an unfamiliar
transplant but sustains the fungal community in
native soils (Broeckling et al., 2008), and this process
was partly mediated by root exudates. In this study,
we demonstrate that one ABC transporter mutant,
abcg30, significantly affects the soil microbial com-
munity compared with the wild type. The function of
abcg30 (also known as Atpdr2) is unknown; however,
the organ-specific expression pattern of this gene
shows that it is highly expressed in root epidermal
cells (Birnbaum et al., 2003; Badri et al., 2008). In
plants, the pleiotropic drug resistance gene family
has been shown to be involved in extruding the
antifungal diterpene from leaves (Jasinski et al.,
2001), heavy metal detoxification (Lee et al., 2005),
herbicide detoxification (Ito and Gray, 2006), and
nonhost resistance (Stein et al., 2006). Although
abcg30 caused the most dramatic changes in overall
microbial composition in the soil compared to the
wild type, it should be noted that the other ABC
transporter mutants tested in this study generated
changesincertainmicrobialOTUs(datanotshown).
The changes observed in soil microbial diversity
between the generations could be due to a direct plant
effect or to soil microbial population dynamics. Based
on our results and experimental design, we conclude
that the observed changes in soil microbial diversity
are due to the effect of the plant because (1) no
microbial changes in the soil were observed in the
control (no plant) from the first to second generation,
and (2) significant changes in soil microbial diversity
from the first to second generation were observed in
abcg30 grown soil but not in the wild type. Additional
studies are needed to determine the longevity of the
abcg30-induced microbial community changes or if
the other mutants may eventually significantly alter
the microbial community.
We further analyzed the root exudates profiles
of seven ABC transporter mutants using two-
dimensional NMR analyses and found that abcg30
has increased phenolics and fewer sugars compared
to the wild type. This result is not in agreement with
the previous report by Badri et al. (2008) where abcg30
root exudates did not show significant differences
with the wild type and the other ABC transporter
Figure 5. Relative abundance of fungal OTUs (1% dissimilarity) sig-
nificantly down- (A) or up-regulated (B) in the abcg30 mutant soils
compared to the wild type. Taxonomic assignments were made based
on the nearest neighbor in GenBank (homology .80%) using a single
representative sequence for each OTU. Col-0, black bars; abcg30, gray
bars.
ABC Transporter Mutant Alters Natural Soil Microbiota
Plant Physiol. Vol. 151, 2009 2011

mutants based on PCA analysis. This observed dis-
crepancy is because in Badri et al. (2008), root exu-
dates were extracted with water and analyzed by gas
chromatography-mass spectrometry to study the dif-
ferences predominantly in the composition of hy-
drophilic compounds such as primary metabolites.
However, in this study, the root exudates were ex-
tracted with ethyl acetate and analyzed by NMR to
study the differences primarily of hydrophobic com-
pounds and hence predominantly in the secondary
metabolites between the mutants and the wild type. In
both studies, the contributing factors (primary versus
secondary metabolites) for PCA analyses were differ-
ent, which explains why abcg30 root exudates were
significantly different from the wild type and other
mutants used in this study.
As stated above, a wide variety of compounds
exhibited an altered level of secretion in abcg30; how-
ever, this does not necessarily mean that secretion of
all of these compounds is under the direct control of
ABCG30. Instead, the pleiotropic effect of the single
gene (abcg30) mutation may be linked to the coreg-
ulation of metabolic processes or other transport
systems. Both possibilities were evident in our whole-
genome expression analyses, which showed that the
genes involved in secondary metabolite biosynthesis
and transporting systems (lipid transporters and ABC
transporters) are up-regulated in abcg30 but not in the
wild type. Conversely, we observed that sugar and
mannitol transporters were down-regulated in abcg30
compared with the wild type due to the pleiotropic
effect of the gene mutation. Because we observed
relatively more salicylic acid and sinapic acid in the
root exudates of abcg30 compared with the wild type,
the use of other gene mutations like sid2 (salicylic acid
deficient) and fah1 (sinapate deficient) may provide
insight about the role of salicylic or sinapic acids in
regulating soil microbial composition. In this study, we
used the tt4 (defective in flavonoid synthesis) mutant
but found no significant effect in reshaping soil fungal
or bacterial communities compared to the wild type
(Supplemental Tables S1 and S2). These data suggest
that flavonoids are not common, or widespread, sig-
nals regulating the overall soil microbial community,
despite their ability to induce nodulation of legumes
by Rhizobium (Peters et al., 1986).
In vitro serial dilution plating showed that abcg30
root exudates significantly reduced total microbial
growth (i.e. colony-forming units). From this experi-
ment, it is impossible to determine whether the lack of
sugars, increase in phenolics, or both were responsible
for the reduced microbial growth; however, like the
study of Broeckling et al. (2008), this experiment
provides additional proof of a direct link between
root exudates and soil microbial growth and survival.
Pyrosequencing analyses revealed that the abcg30
soil microbial community is enriched with rhizobac-
teria that may be related to heavy metal remediation,
detoxification of toxic chemicals, and nitrogen fixation.
Unfortunately, the pyrosequencing analysis as em-
ployed here was semiquantitative and could not be
used to accurately estimate the absolute abundance of
specific microbes or OTUs. However, it is clear that the
abcg30 soil microbial community is shifting and that
the relative abundance of several OTUs closely related
to known PGPRs or otherwise beneficial bacteria is
increasing. Additional studies are needed to deter-
mine the absolute abundance of these OTUs (e.g.
species-specific quantitative PCR) and to what degree
these community shifts may affect such processes as
nitrogen fixation and/or heavy metal remediation.
We hypothesize two possible explanations for the
ability of abcg30 to cultivate microbes related to heavy
metal remediation and detoxification of toxic chemi-
cals: (1) these microbes are especially adept at utilizing
and/or neutralizing the phenolic compounds present
in the abcg30 root exudates, and/or (2) the increase in
phenolics in the exudates have antimicrobial activity
against many of the other bacterial species found in
wild-type-grown soil. Both possible explanations are
evident in our experimental results. Many bacterial
species involved in heavy metal remediation and toxic
chemical(s) decontamination can readily utilize phe-
nolic compounds as substrates (Grech-Mora et al.,
1996; Kaplan and Kittis, 2004; Vainberg et al., 2006;
Kane et al., 2007; Kim et al., 2008), such as those
secreted by abcg30. It is also evident based on the in
vitro soil dilution plating results that abcg30 root
exudates support a less abundant total microbial com-
munity than wild-type root exudates, potentially re-
ducing antagonistic microbe-microbe interactions,
which act to suppress some of the OTUs in the wild-
type soils. Some of the microbial changes might not be
the result of direct interactions with the plant but due
to its direct influence on other members of the soil
community. Further studies are needed to know how
the native soil microbe-induced root exudate profiles
of the wild type and abcg30 alter the total soil microbes
at the community level.
We also reported that abcg30 appears to culture
Xyllela sp. and Mycobacterium sp. compared to the wild
type. Both of these microbes could be related to
important plant or human pathogenic bacteria, respec-
tively, and thus deserve further identification. It is
likely that the wild type does not support the growth
of these bacteria because of no particular interactions
with the plant; however, the alteration of the ratio of
root exudates might inadvertently promote the growth
of these bacteria in the soil cultured by the mutant.
Thus, putative compounds in the exudates of the wild
type could potentially be used as antimicrobials spe-
cific for these otherwise pathogenic bacteria.
Overall, our results show that abcg30 and the resul-
tant ratio and composition of both phenolics and
sugars in the root exudates have a profound effect on
natural soil microbial composition. This is the first
study that explicitly shows how changing the blend of
rhizosphere chemicals can lead to changes in microbial
composition. In addition, this study shows that spe-
cific mutations in genes involved in root exudation of
Badri et al.
2012 Plant Physiol. Vol. 151, 2009

phytochemicals, such as ABC transporters, present in
otherwise functional plants can have significant effects
on soil microbial composition, thus strengthening the
notion that the function of certain genes might not be
restricted to intrinsic plant physiology but to interac-
tions with the environment. As such, we believe that
this work provides a strong foundation for the devel-
opment of new technologies that exploit ABC trans-
porters’ control of root exudation to modify the soil
microbial community composition for beneficial pur-
poses.
MATERIALS AND METHODS
Soil Experiment
For performing the soil microbe diversity experiment, we followed the
method described by Broeckling et al. (2008) with slight modifications. We
used Illinois soil collected from under Arabidopsis (Arabidopsis thaliana) plants
for the soil microbe diversity experiment. The soil was collected in 2007
(collected by Joy Bergelson, University of Chicago, and described in detail in
Broeckling et al., 2008) at 42°05#34$N, 86°21#19$W, elevation 630 feet. The
collected field soil was shipped to Fort Collins, Colorado, in air-tight coolers
and stored in a cold room (4°C) until further use. The soil was air-dried,
cleaned of plant debris, homogenized by hand, and transferred into pots (9 3
9312-cm pots). The bottom of the pots were lined with Whatmann 3MM filter
paper to avoid soil loss. The pots were moved to a greenhouse bench and
watered sufficiently (two or three times a week) for 3 weeks, during which the
soil’s existing seed bank seedlings were continuously removed. After the
complete removal of the existing seed bank seedlings, we sowed surface-
sterilized Arabidopsis seeds. Pots were maintained in a greenhouse under
ambient conditions for optimum plant growth. For each mutant (and Col-0),
nine replicate pots were maintained. The pots without plants served as
negative controls to see the environmental effects contributing to the changes
in soil microbial community structure. The aerial portions of the plants were
harvested after 10 weeks for each generation, the tissue was dried for 3 d at
70°C, and the dry weight biomass was recorded. A 2- to 3-week dormancy
period (no watering) was applied between each generation to allow the root
systems of previous plants to die. The first generation was seeded in August
2007 and harvested in October 2007, and the second generation was seeded in
December 2007 and harvested in February 2008. A list of Arabidopsis mutant
lines and their parental background used in this experiment is presented in
Supplemental Table S6.
Soil Sampling
The top 2.0 cm of soil within a 0.7-cm radius around the crown of the plant
was sampled using a cork borer sterilized with bleach and rinsed thoroughly
with distilled water. This sampling procedure was adopted to allow for
multigenerational sampling without significantly disturbing the soil texture
between the generations. Soil samples were transferred into scintillation vials
and stored at 220°C until processing. For the ARISA analyses, we pooled the
soil samples collected from three different pots of each Arabidopsis line for a
total of three replicates per treatment.
Soil DNA Extraction
To characterize the soil microbial community, total DNA was extracted
from soil and amplified by PCR using internal-transcribed spacer (ITS)-
specific primers, and the amplified products were sequenced to identify taxa
level. Briefly, DNA was extracted from the soils using a MoBio ultraclean soil
DNA kit (Mo Bio) according to the manufacturer’s instructions except for the
addition of an extra ethanol wash. The DNA was quantified using a Nanodrop
spectrophotometer (Nanodrop Technologies) and all DNA had a A260:A280
ratio between 1.7 and 1.9.
ARISA
PCR amplification was performed with the fungal-specific 2234C and
3126T primer set (Sequerra et al., 1997) or the bacterial-specific ITSF and
ITSReub primer set (Cardinale et al., 2004). PCR reactions contained 5 mL(10
ng mL21) soil DNA, 10 mL23jumpstart reaction mix (Sigma-Aldrich), 2.4 mL
25 mMMgCl2, 0.2 mL1mMfluorescein, and 0.4 mL10mMforward and reverse
primers and were brought to 20 mL with deionized water. The PCR products
were amplified for 30 cycles (at 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s).
PCR reactions were diluted with 80 mL of distilled water, and a 2-mL aliquot
was added to 10 mL loading buffer (1,250 mL formamide and 50 mL Genescan
2500 [TAMRA] size standard) and analyzed directly by capillary electropho-
resis (ABI Prism 310; Applied Biosystems) without further modification, i.e.
denaturation heating. Electrophoresis conditions were as follows: 47-cm
capillary, Genescan POP4 polymer, 15-s injection for 15 kV, and 45-min
electrophoresis at 15 kV. Scoring of amplicons into unique bins was performed
using Genemapper software (version 4). Species richness of each sample was
determined as the number of amplicons above a threshold of 50 relative
fluorescence units with the understanding that any given peak may contain
amplicons from multiple species (Manter and Vivanco, 2007).
Pyrosequencing Analyses
Amplification of the fungal ITS or bacterial (16S) rRNA genes were
performed with the following primers:
fungi, 2234C, 5#-gcctccctcgcgccatcagGTTTCCGTAGGTGAACCTGC-3#
and 3126T, 5#-gccttgccagcccgctcagATATGCTTAAGTTCAGCGGGT-3#; bacte-
ria, 27F, 5#-gcctccctcgcgccatcagAGAGTTTGATCMTGGCTCAG-3#and 388R,
5#-gccttgccagcccgctcagTCTGCTGCCTCCCGTAGGAGT-3#.
The lowercase and underlined regions of the above primers are necessary
adapters for binding and amplification using the pyrosquencing process, and
the uppercase regions are primers targeted to conserved regions of the rRNA
genes. The fungal primers listed here were previously employed in several
fungal diversity studies (Sequerra et al., 1997; Ranjard et al., 2001; Lejon et al.,
2005; Broz et al., 2007) and have clearly demonstrated an ability to amplify
fungi belonging to Ascomycota, Basidiomycota, Zygomycota, Oomycota,
Chytridiomycota, and Plasmodiophoromycota, while not amplifying bacteria
or plant DNA (Ranjard et al., 2001). The bacterial primers were demonstrated
to amplify a wide variety of bacteria (Lane, 1991; Marchesi et al., 1998);
however, we must include the caveat that no single combination of rRNA
primers can amplify all fungal or bacterial isolates (Brunk et al., 1996). PCR
reaction conditions were the same as outlined above for the ARISA analysis.
Following PCR, the products were visually checked on an agarose gel, and
each successful reaction was purified using AMPure beads (Agencourt).
Purified PCR amplicons, from the three replicate soil samples per treat-
ment, were pooled at a ratio of 4:1 v/v (bacteria:fungi). We chose this pooling
procedure, as opposed to combining all reactions on an equimolar ratio,
because an accurate determination of the number of molecules in the fungal
amplicons is impossible due to length heterogeneity in the ITS1 region. The
pyrosequencing was performed under contract with the University of Florida
Genomics Facility using 1/16 of a PicoTiter-Plate (454 Life Sciences) yielding a
total of approximately 8,000 sequence reads.
In order to minimize the effects of random sequence errors, we removed all
sequences with multiple undetermined residues (n.3) or a single primer
nucleotide mismatch. The resulting sequences, after removal of the unique
barcode, averaged 275 bp in length. Because sampling effort (i.e. number of
sequence reads) can influence estimates of total species richness, a subset of
160 fungi and 2,489 bacterial sequences were randomly selected and used to
create a single library for each of the Atabcg30 and wild-type soils. A multiple
sequence alignment was performed individually for each of the fungal (n=
320) and bacterial (n= 4978) libraries using MUSCLE (parameters set to
-maxiters 2; Edgar, 2004). From this alignment, a distance matrix was
constructed using DNADIST (Jukes-Cantor correction) from PHYLIP version
3.68 (Felsenstein, 1989, 2005). The distance matrix was then input into DOTUR
(Schloss and Handelsman, 2005) for clustering the sequences into OTUs based
on the genetic distance between sequences and the generation of rarefaction
curves for making estimates of the total OTU richness (ACE and Chao1; Chao
and Lee, 1992; Chao et al., 1993) and diversity (Shannon-Weaver Index;
Magurran, 1988) indices. Community similarity was determined using
E-libshuff (Schloss and Handelsman, 2006), which uses a Monte Carlo testing
procedure to evaluate differences between each of the communities (Schloss,
2008).
A more detailed analysis of the difference between the DOTUR assigned
microbial OTUs (1% dissimilarity) in the abcg30 and wild-type soils was
conducted as follows: for each OTU, the probability that the OTU relative
abundance differed between the abcg30 and wild-type soils was estimated
ABC Transporter Mutant Alters Natural Soil Microbiota
Plant Physiol. Vol. 151, 2009 2013

using the statistical test developed by Audic and Claverie (1997). For the
bacterial OTUs, taxonomic assignments were made with the ribosomal
database project’s naı
¨ve Bayesian classifier (80% confidence threshold) using
a single representative sequence from each OTU (Wang et al., 2007). For the
fungal OTUs, taxonomic assignments were made based on the nearest
neighbor in the GenBank nonredundant database (homology $80%) using a
single representative sequence from each OTU. All reported taxonomic
assignments are the lowest identified taxonomic level with a confidence
threshold (bacteria) or homology (fungi) $80%. Homology (H) was calculated
using the following equation:
H¼
I2G
L3100
where L= length of query sequence, I= identities, and G= gaps from the
default BLAST output.
Plant Material and Growth Conditions
Arabidopsis seeds were surface-sterilized with bleach for 1 min followed
by five rinses in sterile distilled water and plated on Murashige and Skoog
(MS; Murashige and Skoog, 1962) salts supplemented with 3% Suc and 0.8%
Bacto agar in petri dishes. Plates were incubated in a growth chamber
(Percival Scientific) at 25°C with a photoperiod of 16 h light/8 h dark for
germination. To collect root exudates, 7-d-old seedlings were transferred to
six-well culture plates (VWR Scientific), with each well containing 5 mL of
liquid MS (MS basal salts supplemented with 1% Suc), incubated on an orbital
shaker at 90 rpm, and illuminated under cool-white fluorescent light (45 mmol
m22s21) with a photoperiod of 16 h light/8 h dark at 25°C. According to
previously published methods (Loyola-Vargas et al., 2007; Badri et al., 2008),
when plants were 18 d old, they were washed with sterile water to remove the
surface-adhering exudates and transferred to new six-well plates containing 5
mL MS liquid media and incubated on an orbital shaker at 90 rpm and
illuminated under cool-white fluorescent light (45 mmol m22s21) with a
photoperiod of 16 h light/8 h dark at 25°C. The exudates were collected 3 d
after transfer. For each replicate analysis, we collected 1500 mL exudates from
300 individually grown Arabidopsis plants. Root exudates were collected for
the wild type and mutants from two independent experiments in triplicate.
Extraction of Phytochemicals
Three days after the transfer described above, the collected liquid media
were filtered through nylon filters of 0.45-mm pore size (Millipore) prior to
freeze drying (Labconco). The freeze-dried powder was dissolved in 10 mL of
distilled water and partitioned three times with an equal volume of ethyl
acetate (EtOAc; Fisher Scientific). All three EtOAc fractions were pooled, and
the remaining water residues were removed using sodium sulfate as a drying
agent. The dried concentrate was dissolved in 800 mL of methanol for
subsequent NMR analysis.
NMR Experiments
EtOAc extracts of Arabidopsis root exudates were dissolved in 1 mL of
50% MeOH-d4in buffer (90 mMKH2PO4, pH 6) containing 0.05% w/v
trimethyl silyl propionic acid sodium salt (TMSP). The mixture was vortexed
at room temperature for 30 s, ultrasonicated for 1 min, and centrifuged at
30,000 rpm at 4°C for 5 min. NMR spectra were acquired at 25°C on a 600-MHz
Bruker AV-600 spectrometer equipped with a cryoprobe operating at proton
frequency of 600.13 MHz. MeOH-d4was used as the internal lock. Each
1H-NMR spectrum consisted of 256 scans requiring 8 min and 30 s acquisition
time with the following parameters: 0.12 Hz/point, pulse width of 30°(11.3
ms), and relaxation delay of 2 s. A presaturation sequence was used to
suppress the residual water signal with low power selective irradiation at the
water frequency during the recycle delay. Free induction decay was Fourier
transformed with a line-broadening factor of 0.3 Hz. The resulting spectra
were manually phased, baseline corrected, and calibrated to the internal
standard TMSP at 0.0 mLL
21using Topspin (version 2.1; Bruker). Two
dimensional J-resolved NMR spectra were acquired using 16 scans per 64
increments for F1 and 1,638.4 k for F2 using spectral widths of 7239.4 Hz in F2
(chemical shift axis) and 50 Hz in F1 (spin-spin coupling constant axis). A 1.5-s
relaxation delay was employed. Data sets were zero-filled to 512 points in F1,
and both dimensions were multiplied by sine-bell functions (SSB = 0) prior to
double complex FT. J-resolved spectra were tilted by 45°, aligned about F1,
and then calibrated to TMSP using Topspin. In order to obtain projected
spectra, the signals of two-dimensional J-resolved spectra were summed up to
the F2 direction by Topspin. The COSY spectra were acquired with a 1.0-s
relaxationdelay and 6,009.6 Hz spectral width in bothdimensions. The window
function for the COSY spectra was Qsine (SSB = 2.0). The HSQC spectra were
obtained with a 1.0-s relaxation delay and 6,009.15-Hz spectral width in F2 and
164 Hz in F1. The HMBCspectra were recordedwith the same parametersas the
HSQC spectrum except for 31,692.7 Hz of the spectral width in F2.
NMR Data Analysis
Spectral intensities of 1H-NMR spectra were scaled to the total intensity
and reduced to integrated regions of equal width (0.04) corresponding to the
region of d0.3 to d10.5 using AMIX (version 3.8; Bruker). The regions of d4.7
to d5.0 and d3.28 to d3.40 were excluded from the analysis because of the
residual signal of water and methanol. PCA was performed with the SIMCA-P
software (version 11.0; Umetrics). The scaling method for PCA was Pareto.
Matching of two-dimensional J-resolved signals was performed by AMIX
using automatic noise level.
Soil Microbe Screening
Viable soil microbe screening was performed by supplementing microbe
cultures with plant root exudates to check whether root exudates have any
effect on microbes in vitro. Arabidopsis-adapted soil planted with wild-type
Arabidopsis was serially diluted with sterile distilled water and spread onto
soil extract agar (SEA) plates (Alef and Nannapieri, 1995) for bacterial
enumeration or SEA plates amended with streptomycin (50 mg/mL) and
ampicillin ((50 mg/mL) for fungal enumeration. Before spreading onto SEA
plates, root exudates (1:5, v/v) collected from the wild type (Col-0), abcg30,or
a control (MS liquid media) were added to the serial dilutions and incubated
for 16 h at 26°C. Colony-forming units of fungal and bacterial colonies were
counted after 24- and 48-h incubations at 26°C. Root exudates were collected
by growing wild-type and abcg30 plants in MS liquid media for 30 d on an
orbital shaker at 90 rpm and illuminated under cool-white fluorescent light (45
mmol m22s21) with a photoperiod of 16 h light/8 h dark at 25°C. The exudates
were collected, filtered through nylon filters of 0.45-mm pore size (Millipore;
Durapore membrane filters) prior to freeze drying (Labconco). The freeze-
dried powder was dissolved in distilled water and filter-sterilized through
syringe filters before supplementing into soil serial dilutions.
Statistical Analyses
We used nonparametric multidimensional scaling (NMS) to examine
bacterial and fungal communities in the rhizospheric soil samples of Arabi-
dopsis wild-type and ABC transporter mutants. NMS is an interactive best-fit
ordination technique that arranges samples so that the distance between soil
samples in ordination space is in rank order with their similarities in
community structure (Clarke, 1993). The Sorenson distance metric was used
as a measure of dissimilarity in all NMS ordinations (Faith et al., 1987;
McCune and Grace, 2002). We also used MRPP to test the null hypotheses of
no difference in the microbial communities of all soil samples included in this
study. MRPP is a nonparametric, multivariate method used to make statistical
comparisons among two or more groups (Zimmerman et al., 1985). The P
value associated with the MRPP test statistic describes how likely an observed
difference is between the groups.
Microarray Analyses
We used 20-d-old plants raised in liquid culture as described in this study
for total RNA extraction from root tissues of both the wild type and abcg30 by
using Trizol reagent following the manufacturer’s instructions. cRNA was
prepared following the manufacturer’s instructions (www.affymetrix.com/
support/technical/manual/expression-manual.affx). The Affymterix micro-
arrays (Arabidopsis ATH1 genome array) containing 22,810 probe sets
representing approximately 80% of the gene sequences on a single array
was used in this study. Labeling and hybridization on the ATH1 microarrays
(one sample per chip) was performed according to the manufacturer’s
instructions (www.affymetrix.com/support/technical/manual/expression-
manual.affx). The labeling and hybridization analyses were performed at
Yale University Microarray facility center (Keck Biotechnology Resources
Badri et al.
2014 Plant Physiol. Vol. 151, 2009

Laboratory). The probe arrays were scanned and further analyzed with
GENESPRING software (version 5.0; Silicon Genetics). Normalization per
gene and per chip of the log2values was performed to allow the comparison of
two independent biological replicates. In addition, normalization was per-
formed separately for each biological replicate using the flags (“present,”
“marginal,” or “absent”) assigned by Affymetrix treatment of the arrays. Such
a procedure allows eliminating the transcripts with very low signals in both
treatments followed by multiple hypotheses testing to generate Pvalue. Data
were analyzed for each of the Atabcg30 replicates with corresponding wild-
type replicates. The genes that reveal significant changes (P= 0.01) in their
expression were considered. Moreover a cutoff value of 1.5-fold change was
adopted to discriminate expression of genes that were differentially altered in
Atabcg30 compared with the wild type. Annotation of the genes represented
on the microarray to genomic open reading frames was done with “gene
description” and “gene ontology” programs of GENESPRING based on the
information from the International Arabidopsis Genome Initiative sequencing
project in collaboration with The Institute for Genomic Research.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Comparative analysis of relative abundance
(peak height) of fungal (A) and bacterial (B) phylotypes significantly
differed between wild type (Col-0) and abcg30.
Supplemental Figure S2. 1H NMR (A), 2D J-resolved (B), and projected 1D
J-resolved (C) spectra of control Arabidopsis root exudates (Col 0) in the
range of d6.0 to d8.7.
Supplemental Figure S3. Loading plot of PC1 (A) and loading plot of PC2
(B) of principal component analysis of Arabidopsis root exudates using
projected one-dimensional J-resolved spectrum.
Supplemental Figure S4. Two-dimensional J-resolved spectrum of wild-
type root exudates with compound assignments.
Supplemental Figure S5. Two-dimensional J-resolved spectrum of wild-
type root exudates with compound assignments.
Supplemental Figure S6. Two-dimensional J-resolved spectrum of wild-
type root exudates with compound assignments.
Supplemental Figure S7. Comparison of one-dimensional projected
J-resolved spectra of Arabidopsis wild-type and mutant root exudates
in the range of d8.6 to d5.7.
Supplemental Figure S8. Comparison of one-dimensional projected
J-resolved spectra of Arabidopsis wild-type and mutant root exudates
in the range of d5.6 to d4.3.
Supplemental Figure S9. Comparison of one-dimensional projected
J-resolved spectra of Arabidopsis wild-type and mutant root exudates
in the range of d4.3 to d3.0.
Supplemental Figure S10. Comparison of one-dimensional projected
J-resolved spectra of Arabidopsis wild-type and mutant root exudates
in the range of d3.0 to d0.5.
Supplemental Figure S11. Percentages of genes belonging to differ-
ent functional categories were significantly up-regulated and down-
regulated in abcg30 root tissues compared with wild type (Col-0) by
whole-genome expression analyses.
Supplemental Figure S12. Phylogenetic tree showing the evolutionary
relationships of 66 taxa of the group a-proteobacteria based on rDNA
sequences.
Supplemental Table S1. Multiresponse permutation procedures (MRPP)
analysis comparing the microbial community associated with each
Arabidopsis ABC transporter mutant.
Supplemental Table S2. Pair-wise comparisons of the wild type (Col-0)
from MRPP analysis comparing the microbial community associated
with each Arabidopsis ABC transporter mutant.
Supplemental Table S3. Relative NMR intensity of changed metabolites in
Col-0 and abcg30 using projected J-resolved spectra (signals are normal-
ized to 1,000 of internal standard [TMSP] at d0.0).
Supplemental Table S4. Select list of genes differentially expressed in
abcg30 roots compared with the wild type.
Supplemental Table S5. In vitro analysis of the Arabidopsis wild type
(Col-0), abcg30 root exudates, and MS liquid media (control) effect on
Arabidopsis soil microbes by plate counting assay
Supplemental Table S6. List of ABC transporters and their T-DNA
knockout mutants used in this study.
ACKNOWLEDGMENT
We thank Joy Bergelson for providing soils for this study.
Received September 14, 2009; accepted October 19, 2009; published October
23, 2009.
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ABC Transporter Mutant Alters Natural Soil Microbiota
Plant Physiol. Vol. 151, 2009 2017