Loss of Abaxial Leaf Epicuticular Wax in Medicago truncatulairg1/palm1 Mutants Results in Reduced Spore Differentiationof Anthracnose and Nonhost Rust Pathogens W

SrinivasaRaoUppalapati,a,1Yasuhiro Ishiga,a,1VanthanaDoraiswamy,aMohamedBedair,aShipraMittal,a Jianghua

Chen,a Jin Nakashima,a Yuhong Tang,a Million Tadege,a Pascal Ratet,b Rujin Chen,a Holger Schultheiss,c and

Kirankumar S. Mysorea,2

a Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401b Institut des Sciences du Vegetale, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, Francec BASF Plant Science Company GmbH, D-67117 Limburgerhof, Germany

To identify genes that confer nonhost resistance to biotrophic fungal pathogens, we did a forward-genetics screen using

Medicago truncatula Tnt1 retrotransposon insertion lines. From this screen, we identified an inhibitor of rust germ tube

differentation1 (irg1) mutant that failed to promote preinfection structure differentiation of two rust pathogens, Phakopsora

pachyrhizi and Puccinia emaculata, and one anthracnose pathogen, Colletotrichum trifolii, on the abaxial leaf surface.

Cytological and chemical analyses revealed that the inhibition of rust preinfection structures in irg1 mutants is due to

complete loss of the abaxial epicuticular wax crystals and reduced surface hydrophobicity. The composition of waxes on

abaxial leaf surface of irg1 mutants had >90% reduction of C30 primary alcohols and a preferential increase of C29 and C31

alkanes compared with the wild type. IRG1 encodes a Cys(2)His(2) zinc finger transcription factor, PALM1, which also

controls dissected leaf morphology in M. truncatula. Transcriptome analysis of irg1/palm1 mutants revealed down-

regulation of eceriferum4, an enzyme implicated in primary alcohol biosynthesis, and MYB96, a major transcription factor

that regulates wax biosynthesis. Our results demonstrate that PALM1 plays a role in regulating epicuticular wax metabolism

and transport and that epicuticular wax influences spore differentiation of host and nonhost fungal pathogens.

INTRODUCTION

Rusts are obligate biotrophic foliar pathogens that have evolved

specialized mechanisms of invasion (Heath, 1977). To initiate

rust infection, fungal urediniospores need to adhere to the leaf

surface and subsequently form germ tubes. Germ tubes typically

respond thigmotropically to host leaf surface features, such as

stomata, forming appressoria over these openings (Hoch et al.,

1987). Penetration pegs form at the appressoria-stomata inter-

face andmature into invasive hyphae that invademesophyll cells

eventually differentiating into specialized feeding structures

called haustoria (Heath, 1977; Hoch et al., 1987). Therefore, it

appears that rust pathogens require specific plant surface topo-

graphical and chemical signals to trigger the formation of

preinfection structures (Heath, 1977; Muller and Riederer,

2005). Traditionally, breeding for rust resistance in various crops

mainly relied on host resistance mediated by gene-for-gene

resistance (Ayliffe et al., 2008). However, in many cases, resis-

tance mediated by R genes has little durability in the field due to

the rapid evolution and emergence of new pathogen strains that

can escape recognition by R genes and R gene–mediated

downstream defenses. Alternatively, nonhost resistance (NHR)

is defined as a form of resistance exhibited by an entire plant

species to a particular microbial pathogen and is the most

common and durable form of resistance (Heath, 2000). There-

fore, identification and incorporation of traits that confer NHR to a

broad range of rust fungi is an attractive and durable alternative

to host resistance breeding. However, we know little about genes

that regulate NHR (Mysore and Ryu, 2004).

Asian soybean rust caused byPhakopsora pachyrhiziSydow is

a major concern for soybean (Glycine max) producers in Brazil

and the US (Goellner et al., 2010). Since most of the soybean

cultivars and other economically important legumes are suscep-

tible to soybean rust, there is an increasingly urgent demand for

identification of durable resistance to soybean rust (van de

Mortel et al., 2007). Four single resistance genes to P. pachyrhizi,

Rpp1-4, have been described (Hyten et al., 2007; Garcia et al.,

2008; Silva et al., 2008; Monteros et al., 2010). Interestingly, all

commercial cultivars that are cultivated in the US are susceptible

to rust, and no soybean varieties have been described as having

broad-spectrum resistance to all isolates of P. pachyrhizi

(Posada-Buitrago and Frederick, 2005). Similarly, rust disease

of switchgrass (Panicum virgatum), caused by Puccinia emacu-

lata, is a concern in Oklahoma and other parts of the US and

could become an important factor once switchgrass is grown in

monoculture over a long period of time (Bouton, 2007).

1 These authors contributed equally to this work.2 Address correspondence to ksmysore@noble.org.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Kirankumar S.Mysore (ksmysore@noble.org).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.093104

The Plant Cell, Vol. 24: 353–370, January 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

Therefore, there is an urgent need for identification of novel

genes that could be used for engineering broad-spectrum resis-

tance to soybean rust and/or switchgrass rust isolates.

Identifying novel sources of resistance through large-scale

forward or reverse genetics screens has the potential to improve

crop plant resistance (Heath, 2000; Mysore and Ryu, 2004).

Medicago truncatula is a rapidly emerging model plant species,

especially for legumes. Several genomic tools and resources are

already available for M. truncatula and include an extensive EST

database, genome sequence, gene expression, protein and

metabolite profiling tools, and a collection of insertion and fast

neutron bombardment mutants (Young and Udvardi, 2009). For

large-scale mutagenesis of theM. truncatula genome, a tobacco

(Nicotiana tabacum) retrotransposon, Tnt1, has been introduced

and been shown to efficiently transpose in the M. truncatula

genome during tissue culture, producing insertions that are

stable during seed-to-seed generations (d’Erfurth et al., 2003).

We have now generated ;20,000 Tnt1 insertion lines in M.

truncatulawith an average of 25 insertions per line (Tadege et al.,

2008). We set up a genetic screen to identify M. truncatula

mutants with altered interactions with P. emaculata and/or P.

pachyrhiziwith an aim of identifyingmutants exhibiting enhanced

susceptibility to P. emaculata or P. pachyrhizi. Characterization

of these susceptible mutants would lead to the identification of

target genes for genetic improvement of rust resistance in

switchgrass, soybeans, and other economically important crops,

including wheat (Triticum aestivum) and barley (Hordeum vul-

gare).

The high-throughput forward genetics screen ofM. truncatula

Tnt1 insertion lines performed in this study unexpectedly iden-

tified an inhibitor of rust germ tube differentiation1 (irg1) mutant

that failed to promote preinfection structure differentiation

against nonhost rust fungi, P. emaculata and P. pachyrhizi, and

also to the pathogenic anthracnose fungusColletotrichum trifolii.

Cytological, chemical, and transcriptome analyses revealed that

irg1 is defective in abaxial epicuticular wax deposition and/or

secretion. Flanking sequence tag sequencing of irg1 revealed

that IRG1 encoded a Cys(2)His(2) zinc finger transcription factor

(PALM1) that also controls dissected leaf morphology in M.

truncatula (Chen et al., 2010). Our results unraveled a role for

PALM1 in asymmetric epicuticular wax deposition onM. trunca-

tula leaves, which influences fungal spore differentiation.

RESULTS

Tnt1 Insertional Mutant Screening in

M. truncatula Identified irg1

Initial characterization of the M. truncatula–P. emaculata inter-

action showed that the urediniospores germinate and form germ

tubes on the leaf surface but fail to recognize and form appres-

soria on stomata, thereby precluding successful colonization of

the nonhost plant, M. truncatula (Figure 1A). These interactions

were significantly different from those observed on switchgrass.

On switchgrass leaves, P. emaculata spores adhered, germi-

nated, and formed appressoria possibly after thigmotrophic

signal-mediated oriented growth of the germ tubes to stomata.

The penetrating infection hyphae then formed infection sites that

eventually produced asexual urediniospores. Prehaustorial re-

sistance is a very common form ofNHR to parasitic rust fungi and

is usually mediated by the activation of plant defense responses

(Heath, 1977; Heath, 2000). Interestingly, the M. truncatula NHR

response to P. emaculata was not associated with major tran-

scriptional changes in the phenylpropanoid pathway or other

pathogenesis-related (PR) genes compared with the mock-

inoculated plants (see Supplemental Figure 1 online), suggesting

a passive resistancemechanism. However, it is important to note

that a subtle induction of Chitinase and PR-3 genes was ob-

served at 24 h after inoculation (HAI) when compared with the

wild-type plants (see Supplemental Figure 1 online). To identify

mutants that compromise this particular NHR, we screened 1200

Tnt1 lines or 14,400 independent R0 or R1 plants (12 plants per

each Tnt1 line) for loss of NHR to P. emaculata. Detached leaves

from ;12 plants of each Tnt1 line were challenged with P.

emaculata (see Methods and Supplemental Figure 2 online).

Micro- and macroscopy observations of disease development

were recorded at 8, 24, and 48 HAI and 5 d after inoculation to

identify mutants compromised in NHR.

Strikingly, four independent Tnt1 lines showed inhibition of

germ tube growth and differentiation on the abaxial leaf surface,

instead of the expected phenotype of enhanced susceptibility

(Figures 1A and 1B). We named these mutants irg1 and found

that all of the four identified independent mutants had Tnt1

insertions at different locations in the same exon of one gene.

Therefore, we treated them as null mutant alleles (irg1-1, irg1-2,

irg1-3, and irg1-4). Urediniospores were inoculated on the ab-

axial leaf surfaces (mimics natural infection on host plants), and

the formation of germ tubes and the preinfection structures was

visualized by the green fluorescence emitted by the wheat germ

agglutinin-Alexa Fluor 488 conjugate, a fluorescent lectin that

binds to N-acetyl-glucosamine in the cell walls and thus stains

fungal structures (Figures 1A and 1B). Unlike on the adaxial leaf

surface of the wild-type and irg1-1 mutants where;95% of the

spores adhered, germinated, and formed long germ tubes, on

the abaxial leaf surfaces of irg1-1 mutants, only ;60% of the

spores germinated (Figures 1G and 1H). By contrast, on the

abaxial leaf surface of wild-type plants ;95% of the spores

adhered, germinated, and differentiated into long germ tubes

(Figures 1A and 1H). Intriguingly, the spores that did germinate

on the abaxial leaf surface failed to undergo any further differ-

entiation on irg1 plants and did not showany further growth of the

germ tubes as they did on wild-type plants (Figures 1A, 1B, and

1H). Inhibition of preinfection structure formation on irg1-1 is

most likely due to alterations in surface signal(s) required for the

differentiation of preinfection structures of P. emaculata.

The irg1Mutants Also Showed Inhibited Preinfection

Structure Formation of P. pachyrhizi

To further test if irg1 mutants also inhibit preinfection structure

formation by a broad spectrum of rust pathogens, we inoculated

urediniospores of a direct-penetrating rust fungus, P. pachyrhizi,

on abaxial leaf surfaces of M. truncatula wild-type R108 and

irg1-1 mutants. M. truncatula is an incompatible host to P.

pachyrhizi (Figures 1C to 1F). Although P. pachyrhizi spores

354 The Plant Cell

germinated, formed appressoria, and penetrated the epidermal

cells causing visible necrosis, they failed to sporulate on wild-

typeM. truncatulawhen the inoculated plants weremaintained in

a dew chamber for 24 h with 100% humidity for spore germina-

tion and then incubated in a growth chamberwith low (30 to 40%)

humidity (Figure 1C). Very few necrotic lesions developed on irg1

compared with R108 plants (Figures 1C and 1D). On adaxial and

abaxial leaf surfaces of wild-type R108 plants and adaxial leaf

surface of irg1 mutants, urediniospores adhered, germinated,

and formed germ tubes, and most of the germ tubes underwent

differentiation to form appressoria and directly penetrated the

epidermal cells (Figures 1I to 1K). However, on the abaxial leaf

surface of irg1-1 mutants, the ability of the spores to adhere,

germinate, and form germ tubes with appressoria was severely

compromised, resulting in very low penetration of the epidermal

cells (Figures 1F and 1J). These results further confirmed that the

abaxial leaf surface of irg1 leaves does not promote or inhibit the

formation or differentiation of preinfection structures of at least

two rust pathogens tested.

InhibitionofRustGermTubeDifferentiation in irg1 IsLimited

to Abaxial Leaf Surface

We noticed that the abaxial but not the adaxial leaf surfaces of

irg1-1plantswere glossy in appearancewhen comparedwith the

wild-type R108 plants in which neither surface is glossy (Figures

Figure 1. M. truncatula irg1-1 Mutants Inhibit Preinfection Structure Differentiation by P. emaculata and P. pachyrhizi.

(A) and (B) Confocal micrographs of WGA-Alexa Fluor 488–stained germ tubes (arrows) of P. emaculata on abaxial leaf surfaces of M. truncatula wild-

type R108 (A) and the irg1-1mutant (B). P. emaculata spores germinated and formed germ tubes that grew on the leaf surfaces but failed to differentiate

into appressoria at 72 HAI on wild-type R108, but on irg1-1, spores germinated and the germ tubes failed to grow longer than 50 to 60 mm at 72 HAI.

(C) and (D) Necrotic lesions resulting from direct penetration of P. pachyrhizi on the abaxial leaf surface of wild-type R108 (C) and irg1-1 mutant (D).

(E) P. pachyrhizi spores germinated and formed germ tubes (Gt), and most of them formed appressoria (Ap) within 72 HAI on R108.

(F) By contrast, the germinated spores failed to form long germ tubes on the abaxial leaf surface of irg1-1.

(G) to (K) Development of preinfection structures of P. emaculata and P. pachyrhizi on adaxial and abaxial leaf surfaces of R108, irg1-1, and irg1-2

mutant alleles.

(G) and (H) Preinfection structure formation of P. emaculata on the adaxial (G) and abaxial (H) surfaces of R108 and two independent irg1 alleles (irg1-1

and irg1-2). The percentage of germinated (Ge) P. emaculata urediniospores and differentiated germ tubes without appressoria (Gt) were evaluated as

described in Methods. P. emaculata spores failed to form appressoria or penetrate (Pn) the stomata; therefore, the respective data are not presented.

(I) and (J) Preinfection structure formation of P. pachyrhizi on the adaxial (I) and abaxial (J) surfaces of R108 and two independent irg1 alleles (irg1-1 and

irg1-2). Means 6 SE of 10 replications are presented for each data point in (G) to (J).

(K) Epifluorescence micrographs of the germinated urediniospores of P. pachyrhizi showing different stages of urediniospore differentiation on abaxial

surface of R108, including differentiated germ tubes without appressoria and the germ tubes that formed appressoria (arrow) and successfully

penetrated the epidermal cells (arrow). The number of dead epidermal cells showing autofluorescence resulting from direct penetration (arrow) were

counted to calculate the percentage of penetration (Pn) as described in Methods. Asterisks indicate statistically significant difference evaluated using

paired Student’s t test at P < 0.001.

Bars = 100 mm (A), (B), (E), and (F).

Host Surface Signaling in Rust Infection 355

1C and 1D). This suggested possible alterations in epicuticular

wax loading on the abaxial leaf surface of irg1. In plant interac-

tionswith host-specific biotrophic pathogens, includingErysiphe

pisi andBlumeria graminis, the components of abaxial leaf waxes

were shown to specifically promote the differentiation of pre-

infection structures of E. pisi and B. graminis (Gniwotta et al.,

2005; Hansjakob et al., 2010). To test if two different nonhost rust

pathogens with different preinfection processes require surface

cues for adherence or germ tube differentiation, we examined

the preinfection structure formation of P. emaculata and P.

pachyrhizi on the abaxial and adaxial leaf surfaces of the wild-

type and irg1 plants (Figures 1G to 1J). On the adaxial leaf

surfaces of both wild-type and irg1 plants, almost 90% of the

inoculated urediniospores of P. emaculata that germinated

formed germ tubes with no appressoria on stomata (Figure

1G). Similarly, no inhibition of urediniospore germination or germ

tube elongation was observed on the abaxial side of wild-type

plants (Figure 1H). However, only ;60% of the spores germi-

nated on the abaxial side of the irg1 mutants, and almost all the

germinated spores failed to undergo any further differentiation

(Figure 1H). These results further suggested the absence of

stimulatory signals for germ tube differentiation or presence of

inhibitory signals for growth of P. emaculata on the abaxial

surface of irg1 plants.

Unlike P. emaculata, P. pachyrhizi is a direct-penetrating

biotrophic rust fungus with a broad host range and, based on in

vitro assays conducted on artificial membranes, is suggested not

to require hydrophobic or chemical signals for preinfection struc-

ture formation (Koch and Hoppe, 1988; Goellner et al., 2010). Our

in vivo assays conducted on the adaxial leaf surface showed no

significant differences in germination, appressorium formation, or

epidermal penetration by P. pachyrhizi between wild-type R108

and two independent mutant alleles of irg1 (Figures 1I). Interest-

ingly, P. pachyrhizi showed slightly higher percentage of appres-

soria and penetration rate when inoculated on the abaxial leaf

surface compared with adaxial leaf surface of the wild-type R108

(Figure 1J). However, on the abaxial surface of irg1-1 mutants,

;50%of the spores failed to germinate (Figure 1J). Strikingly, only

;20% of the germinated spores formed appressoria on the

abaxial side of irg1-1 and irg1-2mutant alleles, whereas;75%of

the germinated spores formed appressoria on the abaxial side of

R108 (Figure 1J). Consistent with these results of reduced devel-

opment of infection structures, we observed no induction of any

PR and pathogen-inducible genes on the irg1-1 abaxial leaf

surface inoculated with P. pachyrhizi urediniospores, suggesting

that induced or constitutive defenses were not responsible for the

observed phenotype (see Supplemental Figure 3A online). Fur-

thermore, the total leaf proteins isolated from the irg1 mutants

showedno significant inhibitory effects on the germination or germ

tube differentiation of P. emaculata and P. pachyrhizi uredinio-

spores on plastic (hydrophobic) surfaces, suggesting no predom-

inant antimicrobial protein accumulation in these mutants that

could account for the phenotype we observed on the abaxial

leaf surface (see Supplemental Figures 3B and 3C online).

These results suggested that, instead, the abaxial surface of

irg1 leaves may lack the surface chemical/physical cues re-

quired for the differentiation of preinfection structure formation

by P. pachyrhizi.

irg1 Showed Partial Resistance to C. trifolii but Not to

Phomamedicaginis

To further test if irg1 mutants also exhibit resistance to compat-

ible fungal pathogens, we challenged wild-type R108 and irg1-1

mutants with C. trifolii, a hemibiotrophic pathogen that forms

preinfection structures (Figure 2A), and a necrotrophic pathogen,

P. medicaginis, that directly penetrates without forming appres-

soria (Figure 2B). The percentage of C. trifolii spores that germi-

nated and formed preinfection structures (appressoria) was

significantly reduced on irg1-1mutants, as were the consequent

anthracnose symptoms developed on the abaxial leaf surface

when compared with wild-type R108 (Figure 2A). The percent-

ages of germination and appressoria formation were also slightly

reduced (by ;10%) on the adaxial surface of irg1-1 and irg1-2

mutant alleles compared with the wild-type R108 (Figure 2A).

However, no significant differences in symptom development or

in planta fungal growth of P. medicaginis were observed upon

Figure 2. The irg1Mutant Showed Partial Resistance to C. trifolii but Not

to P. medicaginis.

(A) The percentage of germination (Ge) of conidiospores and germ tubes

with no appressoria (Gt) and germ tubes with differentiated appressoria

(Ap) by C. trifolii conidiospores on the adaxial (Ad) and abaxial (Ab)

surfaces of wild-type M. truncatula R108 and irg1-1 plants. The fungal

structures were stained with lactophenol trypan blue, and the percentage

of spores forming different preinfection structures was evaluated, 72 HAI,

by counting 20 random fields. The data represent the mean of three

independent experiments, and the means 6 SE of 12 replications are

presented for each data point. Means with the same letter within a

particular class (e.g., Ge, Gt, and Ap) are not significantly different using

Duncan’s multiple range test (P < 0.001)

(B) Symptoms (left panel) and fungal growth evaluated by the green

fluorescent protein–tagged P. medicaginis (right panel) following the

inoculation of the spores on the adaxial and abaxial surfaces of wild-type

R108 and irg1 mutant. Bars = 2 cm.

356 The Plant Cell

inoculation on either abaxial or adaxial leaf surfaces of R108 and

irg1-1 (Figure 2B). These results suggested that the effect of irg1

mutants may be limited to those fungi that form preinfection

structures (appressoria) in response to surface signals.

IRG1 Encodes a Cys(2)His(2) Zinc Finger Transcription

Factor and Is Allelic to PALM1

We recovered several Tnt1 flanking sequence tags from the irg1

mutant alleles, and the sequence of one of these had sequence

similarity to a gene that encodes a Cys(2)His(2) zinc finger

transcription factor. Chen et al. (2010) recently showed that the

Cys(2)His(2) zinc finger transcription factor PALM1 controls

trifoliate leaf development in M. truncatula. We decided to

investigate whether there was an association between the irg1

phenotype and the PALM1 mutation. We evaluated four inde-

pendent irg1 alleles identified in this study that have an insertion

in PALM1 and found that irg1-1 is palm1-5 and irg1-2 is palm1-4

(see Supplemental Figure 4 online). In addition, all irg1 mutants

as well as palm1 had five leaflets, unlike a typical M. truncatula

leaf, which has three leaflets (see Supplemental Figure 5 online).

To further confirm if IRG1 gene is allelic to PALM1, we

challenged a previously identified palm1 deletion mutant line

M469 of M. truncatula ecotype A17 (palm1-1) and another

independent Tnt1 line (line NF5022, palm1-6) that has an inser-

tion in PALM1 (Chen et al., 2010) with P. pachyrhizi and P.

emaculata. The formation of preinfection structures of P.

pachyrhizi and P. emaculata was severely impaired on the

abaxial leaf surface of all identified alleles of palm1, indicating

that the loss of function of PALM1 is responsible for the irg1

phenotype (see Supplemental Figures 4 online). Since thepalm1-

6 Tnt1 line and the palm1-1M469 deletion line also inhibited rust

germ tube differentiation, we referred to them as irg1-5/palm1-6

and irg1-6/palm1-1, respectively. We further tested if the irg

phenotype results from the loss of function of PALM1 by

complementing the inhibition of germ tube differentiation phe-

notype of irg1-1/palm1-5 (R108 background) and irg1-6/palm1-1

(A17 background) by expressing wild-type PALM1 under the

control of its native promoter in these mutants (Figure 3). The

complemented lines did not show any inhibition of the rust

preinfection structure formation when compared with their re-

spective mutant lines (Figure 3). Taken together, these results

showed that a gene involved in controlling leaf morphology also

contributes to fungal resistance; therefore, going forward, we

renamed our mutants and addressed them as irg1/palm1.

The irg1/palm1Mutant Affects Abaxial Leaf

Epicuticular Wax

Surface chemical and physical signals are known to affect

adhesion, germination, and differentiation of preinfection struc-

tures in pathogenic fungi (Podila et al., 1993; Kolattukudy et al.,

1995; Uppalapati and Fujita, 2000). Therefore, based on our

pathogen assays and our observation that the abaxial leaf

Figure 3. Expression of the Cys(2)His(2) Zinc Finger Transcription Factor PALM1 in the irg1 Mutant Background Restores the Mutant Phenotype.

(A) Complementation of rust preinfection structure formation of P. pachyrhizi by expression of PALM1 in the irg1-1/palm1-5 mutant (R108 ecotype

background). P. pachyrhizi spores germinated and formed long germ tubes and appressoria within 72 HAI on wild-type R108 and complemented lines

(irg1-1/palm1-5:PALM1) but not on the irg1 plants.

(B) Complementation of rust preinfection structure formation of P. pachyrhizi by expressing PALM1 in the irg1-6/palm1-1 mutant in A17 ecotype

background.

Bars = 100 mm.

Host Surface Signaling in Rust Infection 357

surface of irg1/palm1 is glossy, we speculated that irg1mutants

may have defects in cuticle structure or epicuticular wax depo-

sition. Consistent with our hypothesis, scanning electron mi-

croscopy analyses of air-dried leaf samples showed that irg1

leaves, unlike the wild-type leaves, completely lacked epicutic-

ular wax crystals on the abaxial leaf surface but did have wax

crystals on the adaxial surface (Figure 4). The leaves from the

complemented line (irg1-1:PALM1) showed restored wax crys-

tals on the abaxial surface as seen on wild-type leaves.

Epicuticular Waxes of irg1/palm1Mutant Leaves Have Less

Alcohols and More Alkanes

To further understand the nature of compositional changes in wax

(aldehydes and primary alcohols; see Supplemental Figure 6

online) in irg1/palm1, we extracted the epicuticular waxes from

R108, irg1-1/palm1-5, and irg1-2/palm1-4 lines. The total epicu-

ticular waxes isolated from intact leaves were ;1.71-fold higher

per leaf area in R108 compared with irg1 mutants. The amount of

total acids and alcohols in irg1-1/palm1-5 leaveswere 3.2- and2.3-

fold lower, respectively, than theR108 leaves (Figure 5A).However,

the total alkanes were 2.2-fold higher in irg1-1/palm1-5 than the

R108 leaves (Figure 5A). A similar trend in total acids, alcohols, and

alkanes was observed for irg1-2/palm1-4 allele (Figure 5A).

Since our scanning electron microscopy pictures showed a

lack of epicuticular wax crystals on the abaxial surface and the

inhibition of rust spore differentiation was observed only on the

abaxial surfaces, we hypothesized that the abaxial surface of irg1

may either lack a particular wax constituent that is required for

the promotion of germ tube differentiation or may accumulate an

inhibitory factor. To test this hypothesis, we isolated the epicu-

ticular waxes separately from the abaxial and adaxial leaf sur-

faces. The amount of total alcohols, the predominant constituent

ofM. truncatula leafwaxes, and their compositionwere similar on

the adaxial leaf surfaces of wild-type R108 and irg1/palm1

mutants (Figure 5B; see Supplemental Figures 7A and 7B online).

However, on the abaxial surface, significant changes were

observed in the amount and composition of alcohols and alkanes

between R108 and irg1 mutants (Figure 5C; see Supplemental

Figure 7 online). The total alcohols in the abaxial waxes were

;17-fold lower in irg1/palm1 mutant alleles than R108 (Figure

5C). Dramatic reductions of C28 and C30 alcohols in irg1

mutants were the major contributors for ;17-fold reduction in

primary alcohols (see Supplemental Figures 7A and 7B online).

The total alkanes in the abaxial waxes were approximately

threefold higher in irg1 mutants than R108 (Figure 5C). C29 and

C31 alkanes were found in higher amounts on both abaxial and

adaxial surfaces of irg1/palm1 mutant alleles when compared

with R108 leaves (Figure 5C; see Supplemental Figures 7C and

7D online). It is important to note that the total alkanes extracted

from intact leaves using hexane were higher and did not exactly

reflect the total of alkanes from abaxial and adaxial waxes

isolated using gumarabic (Figure 5). It is possible that gumarabic

absorbs alkanes poorly ormost of the epicuticular waxes that are

stripped with gum arabic are predominantly alcohols with some

alkanes. It is also possible that the extracts from the whole leaf

using hexanemight also extract some of the intercuticular waxes

(alkanes). Nevertheless, these results clearly demonstrate that

loss-of-function mutation of IRG1/PALM1 leads to dramatic

alterations in the amount and chemical composition of waxes.

Epicuticular Waxes/Hydrophobicity Promotes Germination

and Appressorium Formation by P. pachyrhizi and

P. emaculata

Our cytological analyses demonstrated that irg1/palm1 mutant

alleles were defective in formation of epicuticular wax crystals

and accumulation of alcohols on abaxial surface of leaves

(Figures 4 and 5). We therefore hypothesized that the compo-

nents of the epicuticular waxes/hydrophobic surface are

Figure 4. Loss of Function of IRG1/PALM1 Results in Loss of Epicutic-

ular Wax Crystal Deposition on the Abaxial Leaf Surface ofM. truncatula.

Scanning electron micrographs showing epicuticular wax crystal struc-

ture on the adaxial (left panel) and abaxial (right panel) leaf surfaces of the

wild-type R108, three independent irg1 mutant alleles (irg1-1/palm1-5,

irg1-2/palm1-4, and irg1-5/palm1-6), and complemented line (irg1-1/

palm1-5:PALM1). Bar = 5 mm.

358 The Plant Cell

required for P. pachyrhizi and P. emaculata to form preinfection

structures. To test this hypothesis, we first quantified the differ-

ences in surface hydrophobicity by measuring the contact angle

at the interface of a drop of liquid (water) with the leaf surface

(sessile drop technique; Curvers et al., 2010). A higher contact

angle (>908) is indicative of poor wetting or a hydrophobic

surface. No significant differences in the contact angle were

observed between adaxial leaf surfaces of the wild-type and

irg1-1 or irg1-2 mutants, which exhibited an average contact

angle of 1408 (Figure 6). However, very distinct differences in

contact angles were observed between the abaxial leaf surfaces

of the wild-type and irg1-1or irg1-2 mutants (Figure 6). The

abaxial leaf surface of wild-type plants exhibited an average

contact angle of 1388, whereas the mutant alleles showed a

dramatic decrease in contact angle (average of 928), which is

indicative of a hydrophilic surface. Although the abaxial surface

of themutants contained dramatically reduced levels of alcohols,

these results suggested that the lack of primary alcohols and

epicuticular wax crystals increased the hydrophilicity of irg1/

palm1 mutant leaves, and the three-dimensional surface mor-

phologies of epicuticular waxes and their polymerization pat-

terns may play an important role in the hydrophobicity of M.

truncatula leaves.

To further test the role ofM. truncatula epicuticular waxes and/

or hydrophobicity contributed by the epicuticular wax in stimu-

lating the differentiation of fungal structures, we made quan-

titative analyses of fungal development (spore germination,

germ tube elongation, and appressorium differentiation) on hy-

drophilic (glass) surfaces, which were uncoated or coated with

Figure 5. Loss of Function of IRG1/PALM1 Results in Dramatically

Reduced Alcohol Content on the Abaxial Leaf Surface of M. truncatula.

Wax content and composition of whole leaf (A) and adaxial (B) and

abaxial (C) leaf surfaces of wild-type and irg1 mutant alleles (irg1-1/

palm1-5 and irg1-2/palm1-4) of M. truncatula. Means 6 SD of six

biological replicates for total waxes (A) and four biological replicates

are presented for abaxial (B) and adaxial (C) waxes for each data point.

Means with the same letter within a particular class (e.g., acids, alcohols,

or alkanes) are not significantly different using Duncan’s multiple range

test (P < 0.001).Figure 6. Loss of Function of IRG1/PALM1 Results in Reduced Contact

Angle and Hydrophobicity of Water Droplets on the Abaxial Leaf Surface

of M. truncatula.

The abaxial leaf surfaces of irg1/palm1 mutants alleles (irg1-1/palm1-5

and irg1-2/palm1-4) are more hydrophilic as visualized by the sessile

water drop method (A) and exhibited lower contact angles (B) when

compared with the wild-type R108 abaxial or adaxial leaf surfaces or

adaxial leaf surface of irg1/palm1. Contact angles presented are the

means (n = 12) 6 SD. Asterisks indicate significant difference evaluated

using paired Student’s t test at P < 0.001.

Host Surface Signaling in Rust Infection 359

epicuticular waxes isolated from soybean, switchgrass, wild-

type M. truncatula, and irg1 mutants. We also studied the effect

of C30 alcohol, the predominant alcohol that was absent on the

abaxial leaf surfaces of irg1/palm1mutants, by quantifying fungal

development on slides coated with C30 alcohol. In addition, we

also studied fungal development on host leaf surfaces where the

waxes were manually removed.

On hydrophilic uncoated glass surfaces mock coated with

hexane, germination and differentiation of P. pachyrhizi uredi-

niospores were severely impaired, wherein only 13.70% (65.13)

of the urediniospores germinated and <5% of the total spotted

spores on the glass slides formed appressoria (Figure 7A). The

glass slides coatedwith total waxes isolated from the adaxial leaf

surface of thewild-typeR108 and irg1/palm1mutants (irg1-1 and

irg1-2) showed a significant increase in percent germination

(53.53 to 70.7%) and appressorium formation (19.15 to 23.59%)

compared with the percentage of germination on the mock-

coated slides (Figure 7A). No significant differences in their ability

to promote spore germination and appressorium formation were

observed between the adaxial waxes isolated from wild-type

R108 and irg1/palm1 mutants (irg1-1 and irg1-2) (Figure 7A).

However, consistent with our observations on the abaxial leaf

surfaces, the total waxes isolated from the abaxial leaf surface of

irg1-1/palm1-5 or irg1-2/palm1-4 promoted only ;30% germi-

nation of the P. pachyrhizi urediniospores compared with the

;70% germination induced by the abaxial waxes of R108

(Figure 7B). Although the total abaxial waxes from irg1 alleles

promoted;30% germination and germ tube growth, they failed

to promote the formation of appressoria, and the percentage of

total spores that formed appressoria was only ;5 to 8%,

comparable to the mock-coated slide glass (Figure 7B). These

results implicated that the physical (hydrophobicity) or chemical

(primary alcohols) cues imparted by the adaxial or abaxial

epicuticular waxes extracted into hexane from the wild-type

plants and adaxial surfaces of irg1mutant alleles can promote P.

pachyrhizi spore germination and appressorium formation.

These results further suggested the requirement of specific plant

signals for fungal development on leaf surfaces.

To further test the specificity of the waxes in promoting P.

pachyrhizi spore germination and differentiation, we evaluated

the effects of total waxes isolated from the abaxial and adaxial

leaf surfaces of soybean, the host for P. pachyrhizi, and switch-

grass, a monocot nonhost of P. pachyrhizi (Figures 7A and 7B).

Although the adaxial and abaxial waxes from soybean promoted

germination comparable to waxes fromM. truncatula R108, they

promotedmore appressorium formation (;40%) comparedwith

the total waxes from adaxial or abaxial surfaces ofM. truncatula

R108 (Figures 7A and 7B). Interestingly, the total waxes from the

adaxial or abaxial surfaces of switchgrass promoted spore

germination that is comparable to R108 or soybean (Figures 7A

and 7B). However, the total waxes from switchgrass leaves failed

to induce a high percentage of appressorium formation; specif-

ically, waxes from the abaxial leaf surfaces of switchgrass

promoted a very low percentage of appressorium formation

(;9%), comparable to the abaxial leaf surface waxes of irg1

mutants (Figures 7A and 7B). These results further suggested

that waxes (or hydrophobicity) in general promote spore germi-

nation but that appressorium formation requires more specific

signals, and the constituents of the waxes may affect these

processes.

Chemical analyses showed that the abaxial surfaces of the irg1

alleles failed to accumulate primary alcohols and form abaxial

wax crystals. Therefore, we tested if the C30 primary alcohol, the

main constituent of M. truncatula waxes, affects the differenti-

ation of spores. Hydrophilic glass slides coated with C30 alco-

hols at 5 mg/cm2 promoted a 3.5-fold increase in percentage of

germination and a 5.4-fold increase in appressorium formation

by the germinating spores of P. pachyrhizi (Figure 8A). The C30

alcohol concentration of 5 mg/cm2 is similar to the concentration

present on M. truncatula leaf surfaces, and stimulation of ap-

pressorium formation was observed even at 0.5 mg/cm2. C30

alcohols also promoted a significant increase in percentage of

germination of P. emaculata spores at high concentration (5 mg/

cm2; Figure 8B). Unlike P. pachyrhizi spores, on hydrophilic

(uncoated glass) surfaces, the urediniospores of P. emaculata

germinated to a higher level (;20 to 30%) and formed germ

tubes that continued to growwithout forming appressoria (Figure

8C). In addition, P. emaculata spores also failed to form appres-

soria on glass slides coated with waxes or primary alcohols

isolated from leaf surfaces (data not shown). Although we were

Figure 7. Effect of Epicuticular Wax on Development of Urediniospores

of P. pachyrhizi and P. emaculata.

Preinfection structure formation by P. pachyrhizi urediniospores, 24 HAI,

on hexane-coated glass slides (mock control) and glass slides coated

with epicuticular waxes extracted from the adaxial (A) or abaxial (B) leaf

surfaces of wild-type M. truncatula (R108) or irg1-1/palm1-5 and irg1-2/

palm-1-4 mutant alleles. Means 6 SE of 12 replications from two

independent experiments are presented for each data point. Means

with the same letter within a group (e.g., percentage of germination [Ge],

long germ tube without appressoria [Gt], and germ tube with appressoria

[Ap]) are not significantly different using Duncan’s multiple range test (P <

0.001). Gm, soybean; Pv, switchgrass.

360 The Plant Cell

unable to test all the different chain length alcohols and other

alkanes or aldehydes, our results clearly suggested that the

primary alcohols present in M. truncatula leaf surfaces provide

the chemical and physical cues for promotion of infection struc-

ture formation by P. pachyrhizi and P. emaculata.

To further confirm the requirement for hydrophobic waxes and

to corroborate the results obtained with the irg1/palm1 mutants

(Figure 1) and glass slides (Figures 7, 8A, and 8B), we followed

the development of P. pachyrhizi urediniospores on the abaxial

surface of the native host plant, soybean (Wax+), and on the

abaxial surface of soybean that was gently rubbed with a buffer

solution containing celite and bentonite to remove most if not all

the epicuticular wax layer (Wax2). On Wax+ abaxial surfaces,

70% of P. pachyrhizi urediniospores formed appressoria (Figure

8D), whereas onWax2 surfaces,;45%of the inoculated spores

formed appressoria and penetrated the epidermal layer (Figure

8D). Consistent with these spore differentiation defects, a sig-

nificant reduction in Asian soybean rust infection was observed

in detached soybean Wax2 leaf surfaces (Figure 8E). It is impor-

tant to note that celite and bentonite remove epicuticular wax

crystals but may also damage the cuticle for a short time (24 h)

and subsequent cuticle-mediated signals are shown not to alter

the local responses to pathogen or the outcome of susceptibility

(Xia et al., 2009). However, we could not completely rule out any

such small contributions of signals in the reduced pathogenicity

on surfaces where the waxes were physically removed. Never-

theless, taken together with the experiments done on glass

slides, these results further confirmed that surface waxes are

critical or function as stimulatory signals for P. pachyrhizi spore

germination and differentiation of appressoria.

Figure 8. Effect of Primary Alcohol and Epicuticular Wax on Development of Urediniospores of P. pachyrhizi and P. emaculata.

(A) Preinfection structure formation (percentage of germination [Ge], long germ tube without appressoria [Gt], and germ tube with appressoria [Ap]) by

P. pachyrhizi urediniospores on glass slides coated with hexane (mock; gray columns) or coated with C30 primary alcohol at 73 10�3 mol/L (;5 mg/mL;

black columns) and 7 3 10�4 mol/L (;0.5 mg/mL; white columns). Means 6 SE of three replications are presented for each data point.

(B) Percentage of germination of P. emaculata urediniospores on glass slides coated with hexane (mock; gray columns), C30 primary alcohol at 7 3

10�3 mol/L (;5 mg/cm2; black columns), and 7 3 10�4 mol/L (;0.5 mg/cm2; white columns).

(C) Germination of urediniospores of P. emaculata on hydrophilic (uncoated glass) surfaces with long germ tubes (arrows), 16 HAI. Bar = 50 mm.

(D) The percentage of appressorium formation on the detached soybean leaves with native (Wax+) or surfaces manipulated to remove the wax (Wax�).Urediniospores of P. pachyrhizi (Pp) were spot inoculated on Wax+ and Wax� abaxial leaf surfaces of soybean, and the number of appressoria was

counted 72 HAI. Similarly, the number of P. emaculata (Pe) appressoria formed on stomata of Wax+ and Wax� abaxial surfaces of switchgrass were

counted 72 HAI. Means 6 SE of 10 replications from two independent experiments are presented for each data point. Asterisks indicate significant

difference evaluated using paired Student’s t test at P < 0.001.

(E) Symptoms (necrosis resulting from direct penetration) induced by P. pachyrhizi urediniospores inoculated on Wax+ and Wax� abaxial leaf surfaces

of soybean, 10 d after inoculation. Bars = 0.75 cm.

(F) P. emaculata formed appressoria on stomata ofWax+ surfaces (arrow) but not on the stomata ofWax� surfaces (arrow) of switchgrass, 72 HAI. Bars = 50mm.

Host Surface Signaling in Rust Infection 361

We further tested the requirement for surface waxes (hydro-

phobicity) for prepenetration development (i.e., germination,

germ tube elongation, and appressorium differentiation) of P.

emaculata urediniospores on Wax+ and Wax2 abaxial surfaces

of the host plant switchgrass. A 35 to 40% reduction in appres-

soria (appressoria formation on stomata) was observed on

Wax2 abaxial surfaces (Figure 8F). On Wax+ switchgrass, the

germinated spores formed appressoria over the stomatal open-

ings (Figure 8F, top panel). On the Wax2 surfaces, although the

germinated spores oriented to recognize the stomata, a signif-

icant number of them failed to form appressoria on the stomata

(Figure 8F, bottom panel). These results along with data about

germination on slides coated with primary alcohols suggested

that P. emaculata spores require waxy surface signals for ap-

pressorium formation and for enhanced germination but not for

initial germ tube growth.

Transcript Profiling Identifies a Role for IRG1/PALM1 in

Regulating Expression of Genes Involved in Long-Chain

Fatty Acid Biosynthesis and Transport

One of the findings of our study is that the loss-of-function

mutation of a transcription factor involved in leaf morphogenesis

impacts epicuticular wax loading in M. truncatula, which in turn

affects germination and differentiation of fungal spores (Figures 4

and 5). To understand this phenomenon at a molecular level, we

compared the transcript profiles of wild-type R108 and three

independent irg1/palm1 homozygous null mutant lines (irg1-1,

irg1-2, and irg1-5) using Affymetrix GeneChip Medicago Ge-

nome Array (Figure 9A). It is important to note that these lines

have Tnt1 insertions in different locations of the same IRG1/

PALM1 exon (see Supplemental Figure 4 online) and also have

multiple insertions in other independent locations in the genome.

Therefore, all threemutants tested had both common and unique

differential gene expression patterns compared with R108 (Fig-

ure 9A, Table 1; see Supplemental Data Set 1 online). We used a

set of 400 upregulated and 48 downregulated genes that were

commonly altered in all threemutant alleles compared with R108

to identify the major pathways targeted by IRG1/PALM1 (Figure

9A, Table 1; see Supplemental Data Set 1 online).

The majority of the most significantly regulated genes (twofold

change, P value < 8.15954E-07), a major portion are predicted to

function in wax/lipid biosynthesis (Table 1; see Supplemental

Data Set 1 online). In irg1/palm1, genes involved in cuticular wax

accumulation, including ECERIFERUM2 (CER2), which encodes

a nuclear-localized protein (Xia et al., 1997), CER1, which en-

codes a putative decarbonylase known to promote long-chain

alkane biosynthesis (Aarts et al., 1995), and several genes

encoding lipid transfer proteins (LTPs) and ABC transporters

were upregulated (more than twofold), whereas most of the

putative wax biosynthetic genes represented on the microarray

chip, including those encoding ECERIFERUM,CER4-2,CER2-2,

CER5 (Pighin et al., 2004), CER8 (Lu et al., 2009), CER6, and

KCS1, were downregulated (Table 1; see Supplemental Data Set

1 online). To further understand the effects ofPALM1mutation on

wax biosynthesis, we studied the expression of M. truncatula

orthologs of Arabidopsis thaliana genes implicated in wax bio-

synthesis using real-time quantitative RT-PCR (qRT-PCR; Table

1, Figure 9B; see Supplemental Table 1 online). Consistent with

themicroarray results, a significant upregulation ofCER2-1 (sev-

enfold) and CER1 (fivefold) was observed in irg1/palm1 (Figure

9B; see Supplemental Table 1 online). Several other genes

implicated in wax biosynthesis, including CER4, which encodes

an alcohol-forming fatty acyl-CoA reductase, CER6-3, CER8,

b-keto acyl-CoA reductase (KCR1 andKCR2), andwax synthase

(WSD1) were downregulated. Approximately fourfold downre-

gulation was observed for CER4-2 and CER6-4 genes, while

others were moderately downregulated (1.5-fold to twofold) in

irg1/palm1 compared with wild-type R108 (Figure 9B; see Sup-

plemental Table 1 online). Interestingly, a homolog of Arabidop-

sis MYB96, a key transcription factor involved in regulation of

wax biosynthesis (Seo et al., 2011), was strongly downregulated

in irg1/palm1 mutant alleles (Figure 9B; see Supplemental Table

1 online). Furthermore, the transcriptome data also identified

several P450 genes and PR genes, including chitinases and b-1-

3-glucanases that were preferentially upregulated in irg1/palm1

Figure 9. Overview of Global Transcript Changes in irg1/palm1Mutants.

(A) Venn diagram representing the overlapping and nonoverlapping

differentially regulated gene sets among three independent irg1 mutant

alleles.

(B) Expression profiles of several genes encoding ECERIFERUM (CER1-4,

6, 8, and 10), PASTICCINO2 (PAS2), b-keto acyl-CoA reductase (KCR1

and KCR2), wax synthase (WSD1), fatty acyl-ACP thioesterase B (FATB),

and MYB transcription factor (MYB30 and MYB96) were evaluated using

qRT-PCR in leaf samples of wild-type R108 and irg1mutant alleles (irg1-1/

palm1-5, irg1-2/palm1-4, and irg1-5/palm1-6) of M. truncatula. Means 6

SE of three biological replications are presented for each data point in

relative to expression of these genes in R108 which was arbitrarily

considered as 1.

362 The Plant Cell

mutants (see Supplemental Data Set 1 online). In summary, the

transcript profiles of the three independent irg1/palm1 alleles

showed alterations in expression of several genes involved in

wax biosynthesis and transport. Functional analysis of the trans-

porters (ABC/LTPs) and overexpression of CER4, CER2, and

MYB96 in M. truncatula will help us to better understand the

mechanism of wax biosynthesis and asymmetric epicuticular

wax loading in irg1 mutants.

DISCUSSION

In this study, we identified and characterized an epicuticular wax

mutant (irg1) ofM. truncatula in a gene encoding aCys(2)His(2)zinc

finger transcription factor and showed that a loss-of-function

mutation of IRG1 causes major changes in epicuticular wax con-

tent and composition on abaxial leaf surfaces. Most intriguingly,

these changes in the irg1 mutant impart resistance to certain

biotrophic fungal pathogens by inhibiting the differentiation of

preinfection structures. Our results showed that irg1/palm1 mu-

tants were completely devoid of wax crystals on the abaxial leaf

surface and suggested a possible role for signals at the cuticle

interface, especially the components of the leaf wax in promotion

or inhibition of rust preinfection structure differentiation on the

abaxial leaf surface of the irg1mutants (Figures 1 and 2). Consis-

tent with our results, two other previous studies have shown

normal development of fungal preinfection structures on the

adaxial surface and short germ tubes with few appressoria on

abaxial leaf surfaces during host-specific pathogenic interactions

involving wild-type pea (Pisum sativum)–E. pisi and wild-type

ryegrass (Lolium spp)–Erysiphe graminis interactions (Carver et al.,

1990; Gniwotta et al., 2005). Variations in wax crystal composition

between adaxial and abaxial leaf surfaceswere implicated in pea–

E. pisi interactions (Gniwotta et al., 2005). However, the genes or

mechanism(s) responsible for this variation in wax crystals be-

tween adaxial and abaxial surfaces were not identified. Further-

more, our results also provided evidence for requirement of waxes

at contact surfaces for appressorium differentiation by P. pachyr-

hizi and further strengthened the earlier hypothesis that surface

composition and hydrophobicity play important roles in appres-

sorium formation and penetration by direct penetrating fungi (Lee

and Dean, 1994).

Interestingly, hydrophobicity was not very critical for germina-

tion or germ tube elongation or preinfection structure develop-

ment by P. emaculata urediniospores. Although primary alcohols

enhanced the percentage germination (Figure 8B), P. emaculata

urediniospores germinated quite efficiently (20 to 25%) and

formed long germ tubes on the hydrophilic (glass) surface (Figure

8C) and on host leaf surfaces where epicuticular waxes were

removed (Figures 8F, bottom panel). However, on the abaxial

Table 1. A List of Selected Wax or Lipid Biosynthesis-Related Genes Differentially Regulated in irg1/palm1 Mutant Alleles (irg1-1/palm1-5, irg1-2/

palm1-4, and irg1-5/palm1-6) Compared with the Wild Type

Probe Sets Putative Gene Annotation P Valuea Fold Change (irg1/Wild Type)b

Mtr.9322.1.S1_at Fatty acid elongase-like protein (CER2-1) 0 7.34

Mtr.34695.1.S1_at LTP-like protein 0 6.49

Mtr.33889.1.S1_at CER1 protein (CER1-2) 3.04922E-34 5.01

Mtr.35178.1.S1_at a-Keto-acid dehydrogenase 0 4.60

Mtr.42509.1.S1_at Arabidopsis gl1 homolog (LTP) 0 4.34

Mtr.34634.1.S1_at Epoxide hydrolase-like protein 0 3.58

Mtr.40882.1.S1_at Aldehyde dehydrogenase 0 3.43

Mtr.13594.1.S1_s_at Lipase-like protein 0 3.25

Mtr.43284.1.S1_at Alcohol dehydrogenase 6.4091E-244 3.13

Mtr.41915.1.S1_at Phospholipase D a1 (PLD a1) 0 3.12

Mtr.44384.1.S1_at PDR7 ABC transporter 3.2127E-124 2.79

Mtr.7160.1.S1_at MRP-like ABC transporter 0 2.02

Mtr.41168.1.S1_at ABC transporter 6.4073E-119 1.96

Mtr.28687.1.S1_at Wax synthase-like protein 1.50875E-41 1.80

Mtr.44147.1.S1_at CER1; unknown 2.51932E-75 1.05

Mtr.32057.1.S1_at CER8 2.14314E-09 0.70

Mtr.12585.1.S1_at b-Ketoacyl-ACP synthetase I (CER6) 0 0.55

Mtr.8997.1.S1_at Enoyl-ACP reductase (CER10) 0 0.55

Mtr.12797.1.S1_at Family II lipase (EXL3) 0 0.39

Mtr.6071.1.S1_s_at MYB96 transcription factor 0 0.38

Mtr.20073.1.S1_at Plant lipid transfer 0 0.36

Mtr.1002.1.S1_at Fatty acid elongase-like protein (CER2-2) 2.7022E-160 0.34

Mtr.26036.1.S1_s_at 3-Ketoacyl-CoA synthase 1 (KCS1) 0 0.33

Mtr.11073.1.S1_at Short-chain dehydrogenase 0 0.24

Mtr.41151.1.S1_s_at Fatty acid elongase-like (CER2-2) 0 0.24

Mtr.38311.1.S1_at Fatty acyl-CoA reductase (CER4-2) 0 0.18

aP value was calculated using associative analysis (Dozmorov and Centola, 2003).bThe fold change presented as irg1/wild type represents the average of the fold changes recorded from irg1-1, irg1-2, and irg1-5 per each respective

gene. A complete list of fold changes in each allele background and the full list of the differentially regulated genes are presented in Supplemental Data

Set 1 online.

Host Surface Signaling in Rust Infection 363

surface of irg1 mutants, although the spores germinated, they

failed to elongate and undergo any further morphogenesis.

These results suggested a possible role for unknown chemical

signals on the abaxial leaf surface of irg1 in inhibiting P.

emaculata germ tube elongation. Plants with defective cuticle

structure and hydrophobicity (Bessire et al., 2007; Chassot et al.,

2007, 2008; Curvers et al., 2010) have strong resistance to

Botrytis cinerea, possibly through increased release of antimi-

crobial compounds from more permeable epidermal cells. Very-

long-chain aldehydes have been shown to promote preinfection

structure formation of B. graminis (Hansjakob et al., 2010) and

Puccinia graminis f. sp tritici (Reisige et al., 2006). Chemical

analyses of irg1 mutants showed increased accumulation of

alkanes in comparison to wild-type R108 (Figure 5; see Supple-

mental Figure 7 online), indicating a possible role in inhibiting the

growth of P. emaculata germ tubes. It is possible that the effects

of other inhibitory signals may be more conspicuous in the

absence of alcohols that show some stimulatory effects on

overall percentage of germination. We tried to isolate any pos-

sible surface antimicrobials (proteins) from leaves using the

methods described for tomato (Solanum lycopersicum) fruit

surfaces (Yeats et al., 2010) but failed to obtain sufficient protein

concentration to conduct spore germination assays. However,

our results using total leaf proteins isolated from irg1/palm1

mutant did not show any inhibitory activity.

The asymmetric distribution of leaf epicuticular waxes to the

abaxial side in the irg1/palm1 mutants is intriguing. Complete

absence of wax crystals on abaxial and adaxial leaf surfaces of

wild-type Arabidopsis (Jenks et al., 1995) and absence of wax

crystals on abaxial but not adaxial surfaces of wild-type Lolium

perenne (Ringelmann et al., 2009) have been reported. Several

studies have reported complex changes in wax composition in

epicuticular wax mutants, including cer1-cer6, resulting from

increased flux of precursors into other metabolic pathways in

Arabidopsis stems (Aarts et al., 1995; Jenks et al., 1995; Rowland

et al., 2006; Kunst and Samuels, 2009). Our chemical analyses

further demonstrated that loss of function of a Cys(2)His(2) zinc

finger transcription factor (PALM1) also results in changes in the

distribution of different classes of waxes among adaxial and

abaxial surfaces. These complex changes in composition and

distribution of waxes in irg1 can be attributed to the increased

flux of metabolites into the decarbonylation pathway (see Sup-

plemental Figure 6 online).

Our transcriptome analysis revealed that IRG1/PALM1 regu-

lates several target genes involved in lipid metabolism and

transport. Based on the complete absence of wax crystals

phenotype on the abaxial side and 50% reduction in primary

alcohols, the major wax component, we expected a dramatic

downregulation of target genes involved in wax biosynthesis.

However, only homologs of CER4 and CER6 showed significant

downregulation (less than or equal to twofold) in irg1 plants

compared with the wild type (Figure 9B, Table 1). Interestingly,

homologs of Arabidopsis CER2 were upregulated in irg1 plants

(Table 1, Figure 9B). CER6/CUT1 encodes a putative KCS that is

potentially involved in elongation of fatty acyl-CoAs longer than

C22, and deletion of CER6/CUT1 results in 93 to 94% reduction

of total wax loading and almost inactive decarbonylation path-

way in Arabidopsis (Millar et al., 1999; Fiebig et al., 2000). CER4

encodes fatty acyl-CoA reductase, which is responsible for

primary alcohol formation in Arabidopsis (Rowland et al., 2006).

Consistent with our chemical analyses, the transcriptome anal-

ysis provided the genetic evidence that the downregulation of a

CER4 was responsible for the reduced alcohols on the abaxial

surface of irg1/palm1mutant leaves. Furthermore,CER1, amajor

enzyme that promotes long-chain alkane biosynthesis (Aarts

et al., 1995; Bourdenx et al., 2011), and homologs of CER1,

including gl1/cer3/wax2 (Rowland et al., 2007; Mao et al., 2012),

were upregulated in the microarray analyses. Taken together

with the chemical analyses, our results also further suggested

that the homologs of Arabidopsis CER4 and CER1 function to

promote alcohol and alkane biosynthesis, respectively, in M.

truncatula. Based on these findings and our results, it is tempting

to speculate that downregulation of CER4 and concomitant

upregulation ofCER1 result in a reduced accumulation of primary

alcohols and increased flux of precursors into the decarbonylation

pathway, resulting in the accumulation of alkanes in irg1mutants of

M. truncatula (Table 1, Figure 5; see Supplemental Figure 6 online).

Therefore, the irg1/palm1mutant may be helpful in elucidating the

biochemical function of CER1 in C30 alcohol biosynthesis in M.

truncatula and other crop legumes. Primary alcohols appear to be

the predominant form of very-long-chain fatty acids in the epicu-

ticular waxes of fabaceae, including M. truncatula (Zhang et al.,

2005, 2007) and pea (Gniwotta et al., 2005). Furthermore, our

results also showed that the plate-type wax morphologies in M.

truncatulamainly contained primary alcohols andwere required for

three-dimensional structure formation of surface waxes and sur-

face hydrophobicity. Our results also suggested that the physical

(hydrophobicity) and chemical surface cues promote spore

germination and germ tube elongation of several biotrophic

fungi. In addition, the physical and chemical surface cues also

promoted appressorium formation by P. pachyrhizi (Figures 7

and 8). Altered wax composition impacts the initial events of

pathogenesis and spore differentiation during compatible plant–

fungal interactions (Kolattukudy et al., 1995; Gniwotta et al.,

2005; Zabka et al., 2008; Hansjakob et al., 2010, 2011). Due to

the absence of the long-chain aldehydes from the leaf cuticular

wax, the glossy11 mutant of maize (Zea mays) doesn’t support

appressorium formation and subsequent prepenetration by B.

graminis and thus is resistant to this fungus (Hansjakob et al.,

2010, 2011). Future studies involving overexpression of CER1

and Myb96 in wild-type and irg1/palm1 mutants may shed new

light on the role of different classes of very-long-chain fatty acids

and their quantities in formation of epicuticular wax structures

and their contributions to hydrophobicity/fungal differentiation in

M. truncatula. The abaxial leaf surface of irg1/palm1 mutants

may also provide a natural leaf surface with altered wax content

and composition that would allow us to test new hypotheses for

the role of epicuticular waxes in fungal spore germination and

germ tube differentiation.

In addition to CER1, CER2 and two genes encoding LTP-like

proteins were upregulated in irg1 mutants when compared with

wild-type R108 (Table 1). Based on the EST information, we

could not confirm if the LTP genes encoded glycosylphospha-

tidylinositol-anchored LTPs. However, one of the LTPs (Affy ID,

Mtr.13293.1.S1_at) showed high similarity to nonspecific LTPs

(ns-LTPs). Some LTPs have been shown to be secreted and

364 The Plant Cell

accumulate extracellularly where they can play a role in diverse

functions, including cuticular wax transport and defense

against pathogens (Segura et al., 1993; Kader, 1996; Kunst

and Samuels, 2003). Loss of function or reduced expression of

a glycosylphosphatidylinositol-anchored LTP results in reduced

alkane accumulation at the plant surface and plays a role in lipid

export (Debono et al., 2009; Lee et al., 2009). Therefore, it is

tempting to speculate that upregulation of the LTP (Mtr.13293.1.

S1_at) could be one of the reasons for increased alkane accu-

mulation in irg1 mutants.

Unlike the contributions of the constituents and morphologies

of the epicuticular waxes, the role of cuticle and cuticular lipids

has been well studied in plant–pathogen interactions. Studies

done with cuticle mutants, including gpat4/gpat8 (Li et al., 2007),

lacs2 and att1 (Xiao et al., 2004; Tang et al., 2007; Lee et al.,

2009), and gl1 (Xia et al., 2010) have shown that these mutants

are more susceptible to pathogens due to a range of alterations,

including stomatal or substomatal spaces or cuticle-derived

active signaling. By contrast, enhanced resistance of att1 and

lacs2 mutants to B. cinerea was reported either via enhanced

perception of the fungal elicitors because of the permeable

surfaces of these mutants leading to the accumulation of anti-

microbials or enhanced upregulation of defense-related genes

(Bessire et al., 2007). Increased susceptibility to the biotrophic

pathogen Erysiphe cichoracearum and resistance to necrotrophic

fungal pathogens B. cinerea and Alternaria brassicicola were

demonstrated in an Arabidopsis rst1 mutant. Interestingly, rst1

was shown to be a cuticular wax mutant with 59.1% reduction in

waxes (and wax crystal deposition) on stem but 43% increase of

waxes in the leaves (Chen et al., 2005). RST1 was shown to

influence plant defense responses by altering the interactions with

jasmonic acid– and salicylic acid–mediated pathways (Mang et al.,

2009). However in our study, irg1/palm1 showed cuticular wax

defects but did not show any alteration in the expression of genes

involved inSApathway or other phytoalexin-mediatedpathways in

mock- or pathogen-inoculated leaves comparedwith thewild type,

suggesting a predominant role of altered abaxial leaf surface

properties (hydrophobicity or wax constituents) in the promotion of

biotrophic fungal differentiation.

In conclusion, we provide evidence for an increased disease

resistance phenotype of irg1/palm1 plants possibly due to al-

tered abaxial leaf surface signals that inhibit differentiation of

fungal preinfection structure in M. truncatula. Although both

developmental and environmental cues affect wax biosynthesis,

only a very few transcription factors that regulate wax biosyn-

thesis have been isolated (Samuels et al., 2008; Kunst and

Samuels, 2009; Seo et al., 2011). Overexpression of the tran-

scription factor WXP1 causes increased accumulation of acyl-

reduction pathway products in M. sativa leaves (Zhang et al.,

2005). Recently, a homeodomain-Leu zipper IV family transcrip-

tion factor was shown to regulate genes involved in cuticle

biosynthesis (Javelle et al., 2010). Furthermore, overexpression of

wax inducer/SHINE family in Arabidopsis and APETALA2 (AP2)/

ethylene-responsive element binding protein–type transcription

factors, WXP1 and WXP2, in Medicago positively regulate wax

biosynthesis (Aharoni et al., 2004; Broun et al., 2004; Zhang et al.,

2005, 2007). Interestingly, Cys(2)His(2) zinc finger transcription

factor (IRG1/PALM1) contains an ERF-associated amphiphilic

repression domain at the C-terminal region and is conserved in

the class II ERF transcriptional repressors of the AP2/ERF

domain proteins (Chen et al., 2010). How a zinc transcription

factor with an ERF transcriptional repressor domain regulates

wax biosynthesis, and asymmetric distribution of epicuticular

wax is a subject for future research. The irg1/palm1 mutant

identified in this study confers altered leaf morphology and wax

deposition on only one side of the leaf surface. However, it is

important to note that the epicuticular waxes are also shown to

regulate nonstomatal water loss and are important in protecting

plants against water loss (Riederer and Schreiber, 2001). Our

preliminary results suggested that irg1 mutants were not more

susceptible to water loss than the wild type, but the effect of loss

of waxes on the abaxial side of the leaf on drought susceptibility

warrants further systematic study. Further characterization of

irg1/palm1 mutants may help to improve our understanding of

asymmetric epicuticular wax loading on leaf surfaces and may

provide us with approaches to specifically engineer adaxial or

abaxial leaf surface waxes to improve fungal resistance both by

regulating fungal differentiation and by improving the penetration

ability of pesticides in agricultural spray applications.

METHODS

Plant Materials

Seeds ofMedicago truncatula cv Jemalong A17, R108, and Tnt1 insertion

lines ofM. truncatulamutant collection, NF0227 (irg1-1/palm1-5), NF1271

(irg1-2/palm1-4), NF1432 (irg1-3), NF4045 (irg1-4), and NF5022 (irg1-5/

palm1-6) in R108 background, and M469 (irg1-6/palm1-1) in A17 back-

ground from the deletion mutant collection ofM. truncatulawere scarified

for 8 min using concentrated sulfuric acid, washed thrice with distilled

water, and germinated on moist filter papers. Two days after germination

in darkness at 248C, 12 seedlings from each Tnt1 line were transferred to

soil (one seedling per cell in 6 3 12 celled trays). Following 3 weeks

incubation in the greenhouse, the plants were transferred to growth

chambers located in a USDA–Animal and Plant Health Inspection

Service–approved BSL2+ facility to conduct soybean rust or switchgrass

(Panicum virgatum) rust inoculation assays.

Screening ofM. truncatula Tnt1 Insertion Population

To identify Tnt1mutants with altered resistance to switchgrass rust, we set

up a forward genetic screen using a detached leaf assay. An isolate of the

switchgrass rust causative agent Puccinia emaculata collected from

Oklahoma (PE-OK1) wasmaintained on a susceptible lowland switchgrass

(P. virgatum cv Summer). Fresh urediniospores of PE-OK1 were collected

using a gelatin capsule spore collector designed by the Cereal Disease

Laboratory, St. Paul, MN, and suspended in distilled water with 0.001%

Tween 20. The abaxial side of the detached leaves were spray inoculated

with 105 spores/mL (0.001% Tween 20) using an artist airbrush (Paasche

Airbrush) set at 2 p.s.i. with a portable air pump (Gast Manufacturing) for

uniform spore deposition. The inoculated leaves were maintained onmoist

filter papers and incubated overnight in dark and then maintained at 248C

with 16-h-light/8-h-dark cycle. It is important to note that we used an R0 or

R1 segregating Tnt1 population for the forward genetic screen, and one

detached leaf from each of 12 plants representing one Tnt1 line was spray

inoculatedwithP. emaculata spores as described above. Tnt1 population is

shown to containmultiple copies of the Tnt1 tag in each line, andmost of the

phenotypes are suggested to segregate in the R1 progeny (Tadege et al.,

Host Surface Signaling in Rust Infection 365

2008). Therefore, we used a sufficiently large segregating R1 population (12

plants per each line) with a possibility of recovering at least one Tnt1 line with

homozygous insertion in a given gene that also confers altered NHR

response to P. emaculata.

Asian Soybean Rust Maintenance and Inoculation Procedures

An isolate of Asian soybean rust pathogen, Phakopsora pachyrhizi, from

Illinois was maintained on the susceptible soybean cultivar (Glycine max

cv Williams) grown in a growth chamber at 228C/198C with a 12-h-light/

12-h-dark cycle (1000 mmol·m–2·s–1). P. pachyrhizi urediniospores col-

lected and prepared as described above for switchgrass rust were used

to inoculate detached leaves or whole plants grown in 72-cell trays. The

inoculated plants were maintained in a dew chamber for 24 h with 100%

humidity maintained at 198C with a 0-h-light/24-h-dark cycle. The plants

were then transferred to a growth chamber (228C/198C with a 12-h-light/

12-h-dark cycle) and incubated further to allow symptom development.

Light, Confocal, and Scanning Electron Microscopy

Initial interactions of P. pachyrhizi or P. emaculatawithM. truncatulawere

recorded by direct observations of inoculated leaves using an Olympus

stereomicroscope (SZX19) or compound microscopes (BX 41) equipped

with fluorescence attachment. For fluorescence microscopy, fungal

mycelia were stained with wheat germ agglutinin (WGA), coupled to the

green fluorescent dye Alexa Fluor 488 (WGA-Alexa Fluor 488; Invitrogen)

as described previously (Uppalapati et al., 2009). Inoculated leaves were

stained with 10 mg/mLWGA-Alexa Fluor 488 by a brief vacuum infiltration

in PBS followed by a 20-min incubation at room temperature. For

microscopy observations, after washing with PBS, whole leaves or

sections of the leaf were placed on a glass slide and mounted using a

cover glass with Dow Corning high vacuum grease for microscopy.

Fluorescence microscopy to document the infection process was done

using an Olympus epifluorescence microscope (BX 41) or a Leica TCS

SP2 AOBS confocal laser scanning microscope (Leica Microsystems)

equipped with 320 (numerical aperture of 0.70) and 363 (numerical

aperture of 1.2, water immersion) objectives using appropriate laser and

excitation filter settings (WGA-Alexa Fluor 488 to 488 nm). Chloroplast

autofluorescence was captured by exciting with the 647-nm line of the

argon-krypton laser and emission detected at 680 nm. A series of optical

sections (z series) were acquired by scanning multiple sections, and the

z-series projections were done with the software provided with the Leica

TCS SP2 AOBS confocal laser scanning microscope.

Scanning electron microscopy analysis of the air-dried leaves was

performed as described previously (Zhang et al., 2005). Briefly, leaves

from the top two internodes were harvested and air-dried at room

temperature in a Petri dish, since the conventional scanning electron

microscopy sample preparation would wash the surface waxes away.

Air-dried leaves were mounted on stubs and coated with;20 nm of 60/

40 Gold-Palladium particles using a Hummer VI sputtering system

(Anatech). Coated surfaces were viewed using a JEOL JSM-840A scan-

ning electron microscope at 15 kV.

Surface Hydrophobicity Measurements

Leaves were fixed to glass slides with double-sided tape and 10-mL

droplets of distilled water were dropped using a micropipette. The

photographs of the droplets were taken, and the contact angle (uC) was

measured using the angle tool incorporated in the image J version 1.44p

software. A total of 12 independent measurements per abaxial or adaxial

leaf surfaces were performed for wild-type and two mutant alleles. Two

water droplets per leaf surface were evaluated on three plants grown in

independent pots from three different experiments. The average of all the

12 replicates was used for contact angle measurements.

Preinfection Structure Formation and Penetration Assays

Approximately 100 spores of P. emaculata or P. pachyrhizi in 10-mL

aliquots were placed on the adaxial or abaxial surface of the detached

leaves from 4-week-old M. truncatula wild-type R108 or irg1 mutant

plants and incubated in dark overnight and then transferred to a growth

chamber (228C/198C with 12-h-light/12-h-dark cycle). To capture the

early stages of preinfection structure formation, 24 HAI, the inoculated

leaves were washed two to three times in a Petri dish with PBS to remove

the free-floating spores and were stained by floating in a PBS solution

supplementedwith 0.05%Tween 20 and 10mg/mLWGA-Alexa Fluor 488

to visualize the fungal germ tubes and appressoria. Urediniospore-

forming germ tubes >60 mm in length were counted as long germ tubes,

whereas germ tubes#50mm in lengthwere counted as short germ tubes.

The number ofP. pachyrhizi andP. emaculata spores that germinated and

formed germ tubes >10 mm in length were evaluated at 24 HAI and were

counted as the percentage of germination. The subsequent develop-

ments were followed 72 HAI, and the germinated tubes forming differen-

tiated appressoria were counted as appressoria, and the differentiated

germ tubes without appressoria that grew on the surface were also

counted from 20 random fields on three independent leaves. On M.

truncatula, the urediniospores of P. emaculata germinated and formed

long germ tubes but failed to form appressoria (on the stomata) and

penetrate. Therefore, only the percentage of germination and the differ-

entiated germ tubes without appressoria were evaluated for P. emaculata

on M. truncatula. The number of dead autofluorescing epidermal cells

resulting fromdirect penetration ofP. pachyrhiziwas counted 72HAI from

20 random fields per each inoculated site and is used to calculate the

percentage of penetration. It is important to note that the percentage of

germination reflects the total percentage of urediniospores that germi-

nated and is a representation of differentiated germ tubes without

appressoria + differentiated appressoria + percentage of penetration.

Fungal Differentiation on Artificial Surfaces and Leaf Surfaces

Manipulated to Remove the Surface Wax Coating

The abaxial and adaxial leaf surface epicuticular waxes from M. truncatula

wild-type and irg1/palm1 mutants, soybean, or switchgrass were isolated

using a polymer film of gum arabic as described (Gniwotta et al., 2005). The

polymer films peeled from two to three leaflets were pooled and extracted

with hexane, evaporated, and resuspended by sonication in fresh hexane.

The hexane solution of waxes from wild-type R108 or irg1 mutants was

adjusted to a final concentration of 0.05mL/cm2 leaf area.Uncoated, frosted

glass slides (253 75mm; VWR International) were coated (three times) with

a hexane solution to cover the whole surface. Hexane was allowed to

completely evaporate between each application as described (Podila et al.,

1993). The C30 alcohol 1-triacontanol (Sigma-Aldrich) was dissolved in

hexane to a final concentration of 73 1023mol/L (;5mg/cm2) and73 1024

mol/L (;0.5 mg/cm2) and was applied to the glass as described above for

the leaf waxes. The approximate coverage per cm2was estimated based on

the assumptions made by Hansjakob et al. (2010). Uncoated glass slides

were coated with same amount of hexane, the solvent used for wax

preparations, and were referred as mock coated/control. To measure the

effect of surface hydrophobicity or surfaces waxes on appressorium forma-

tion, a spore suspension of P. pachyrhizi was applied on the mock control

and glass slides coated with leaf waxes or C30 alcohol.

For removal of epicuticular waxes, detached leaf surfaces of switch-

grass and soybean were gently rubbed with a cotton swab saturated with

a solution containing bentonite (0.02% [w/v]; Sigma-Aldrich) and celite

(1% [w/v]; Sigma-Aldrich) as described (Xia et al., 2009). The uredinio-

spores of P. emaculata and P. pachyrhizi were inoculated on the respec-

tive host leaf surfaces by spotting 10 mL aliquots of 104 spores/mL in

distillated water with 0.001% Tween 20 on the native and surfaces

manipulated to remove wax. The percentage of preinfection structures

366 The Plant Cell

was evaluated as described above in the preinfection structure formation

and penetration assays section.

Infection Assays with Colletotrichum trifolii

C. trifolii race 1 was maintained on potato dextrose agar media (Becton

Dickson and Company). Conidia from 10- to 14-d-old cultures were

harvested, washed in water, and resuspended in sterile distilled water.

Leaves from 4-week-oldwild-typeR108 and irg1mutantswere harvested

and spot inoculated with 10 mL of suspension or spray inoculated with a

suspension containing 1 3 106 spores/mL in 0.005% Tween 20. The

fungal structures were stained with lactophenol trypan blue, and the

percentage of spores forming different preinfection structures (bottom

panel) was evaluated, 72 HAI, by counting 20 random fields. The data

represent the mean of three independent experiments.

Complementation and Stable Transformation

The irg1-6/palm1-6 complementation lines were previously described

(Chen et al., 2010). In this study, we developed stable transgenic lines of

irg1-1 for complementation studies using pCAMBIA3330 carrying a

genomic fragment, including 2.718-kb 59-flanking sequence, 0.756-kb

open reading frame, and 1.028-kb 39-downstream sequence of PALM1

as described (Chen et al., 2010).

Gas Chromatography–Mass Spectrometry Analysis of Cuticular

Wax Content

Total leaf cuticularwax extractionand analysiswasconducted asdescribed

previously (Zhang et al., 2005). Leaf sampleswere collected fromone leaflet

of the top two expanded trifoliates/pentafoliates excised from the major

stems of well-watered wild-type R108 and irg1 mutant alleles. The two

leaflets from the sameplantwere combinedas one leaf sample to represent

one biological replicate. Each sample was added to 10 mL of gas chroma-

tography–mass spectrometry (GC-MS)–grade hexane (Sigma-Aldrich). Tis-

sues were agitated for 2 min, and the solvent was decanted into new glass

tubes. The same amount of hexane was used to rinse the tissues and tubes

for 10 s and was pooled to the sample tube. Hexane was evaporated to

;1mLunder a nitrogen stream, transferred to 2-mL autosampler vials, and

then evaporated completely. The abaxial and adaxial leaf waxes were

extracted as described (Gniwotta et al., 2005). The polymer films from two

leaflets were pooled to represent one biological replicate, extracted with

hexane, and evaporated to 1 mL as described above for the whole leaves.

Six replicates for the whole plant leaf extracts represent six biological

replicates from plants grown in different pots from the same experiment.

The abaxial or adaxial leaf waxes represent the average of four replicates

biological replicates from plants grown in different pots from the same

experiment. These experiments were repeated three independent times,

and data from a representative experiment are presented.

The dried extracts were derivatized using N-methyl-N-trimethylsilyl tri-

fluoroacetamide (+1% trimethylchlorosilane; Pierce Biotechnology) and

were run on an Agilent 7890A gas chromatograph using a splitless injection

as described (Zhang et al., 2005). Quantification was done using the area of

the ions with mass-to-charge ratios of M-15, 117, 57, and 218 for fatty

alcohols, fatty acids, alkanes, and sterols respectively. The amount of total

wax composition and eachcuticularwaxconstituentwas expressedper unit

of leaf area. Leaf areas were determined using a leaf area meter (LI-3000;

Li-Cor). All values represent averages of four biological replicates 6 SD.

Total Protein Isolation and Germination Inhibition Assay

Total protein was extracted from wild-type R108, irg1-1, and irg1-2 M.

truncatula leaves. One gram of leaf tissue was homogenized in buffer (1:2

[w/v]) containing 50mMTris-Cl, pH 7.4, 100mMNaCl, 10%glycerol, 1 mM

phenylmethylsulfonyl fluoride, 10 mM MgCl2, 1 mM EDTA, and complete

miniprotease inhibitor cocktail according to themanufacturer’s instructions

(Roche Diagnostics). Homogenates were centrifuged at 12,000g for 5 min

at 48C to remove debris, and protein concentrations in the homogenates

were determined by the Bradford assay (Bio-Rad). One milliliter of the

urediniospores (23 106 spores/mL) ofP. emaculata and P. pachyrhiziwere

mixed with an equal volume of total protein isolated fromM. truncatulawild

type or irg1mutants at a final concentration of 20mgofprotein/mL, then the

urediniospores were incubated in 6-well cell plastic culture plates (BD

Falcon) for 12 h under dark condition. After incubation, the numbers of P.

emaculata and P. pachyrhizi spores that germinated and germ tubes with

and without differentiated appressoria were counted from five random

fields. The average is used to calculate the percentage of germination and

spores with and without an appressorium.

Statistical Analysis

Significant differences (P# 0.05) within the data sets were tested by one-

way analysis of variance followedDuncan’smultiple range test using SAS

9.2 (SAS Institute). We fitted a general linear model to our factorial data to

estimate the significance of both main effects and interactions for the

cuticular wax content analyses performed using GC-MS (Figure 5).

Similarly, the statistical significances in urediniospore differentiation

were determined using analysis of variance followed by Duncan’s mul-

tiple range test (Figures 2 and 7). Student’s paired t test was used to

analyze the significant differences between wild-type and mutant alleles

or mock and wax treatments (Figures 1, 6, and 8).

Microarray Analyses

To represent different alleles of the irg1 mutant, two independent sam-

pling of three alleles, irg1-1, irg1-2, and irg1-5, totaling six biological

replicates, were used in themicroarray analyses in comparison with three

biological replicates of wild-type plants. All plants were planted in the

greenhouse in separate pots in a single experiment. Total RNA was

purified from leaf tissues of 4-week-old M. truncatula R108 and irg1

mutants using TRIzol reagent (Invitrogen) according to the manufac-

turer’s instructions. Three plants were used for each biological replicate.

Total RNA was extracted from individual plants, and following isolation,

was pooled to represent one biological replicate. The integrity of the RNA

was checked on an Agilent Bioanalyzer 2100, and 10 mg of total RNA was

used as a template for amplification. Probe labeling, chip hybridization,

and scanning were performed according to the manufacturer’s instruc-

tions (Affymetrix). All the raw data were imported into Robust Multichip

Average software and normalized as described (Irizarry et al., 2003). The

presence/absence call for each probe set was obtained from dCHIP

(Fiebig et al., 2000), and differentially expressed genes between sample

pairs were selected using associative analyses, as described (Dozmorov

and Centola, 2003). Genes that showed significant differences in tran-

script levels (twofold or greater and P value < 8.15954E-07) between the

wild-type and mutant plants were selected for further analyses. Type I

family-wise error rate was reduced using the Bonferroni-corrected P

value threshold of 0.05/N, where N represents the number of genes

present on the chip, which is 61,278 forM. truncatula. In this method, the

background noise presented among replicates and technical noise

during microarray experiments was measured by the residual presented

among a group of genes whose residuals are homoscedastic. Genes

whose residuals between the compared sample pairs that are signifi-

cantly higher than themeasured background noise level were considered

to be differentially expressed. Due to the large gene number in the

background measurement, this method enabled Bonferroni correction

without being overly stringent. The data and completeMIAME information

are deposited at ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/)

under accession number E-MEXP-3190.

Host Surface Signaling in Rust Infection 367

Real-Time Quantitative RT-PCR and RNA Gel Blot Analyses

Real-time qRT-PCR was performed as described (Uppalapati et al., 2009)

using thegene-specificprimers (seeSupplemental Table 2online). TotalRNA

was treatedwith TurboDNase (Ambion) to eliminate genomicDNA, and 5mg

of DNase-treated RNA was reverse transcribed using Superscript III reverse

transcriptase (Invitrogen) with oligo(dT)20 primers. The cDNA (1:10) was then

used for qRT-PCR. The qRT-PCRwas performed using Power SYBRGreen

PCRmastermix (AppliedBiosystems) in anoptical 384-well platewith anABI

Prism7900HTsequencedetection system (AppliedBiosystems).Melt-curve

analysis was performed to monitor primer-dimer formation and to check

amplification of gene-specific products. The average threshold cycles (CT)

values calculated from triplicate biological samples were used to determine

the fold expression relative to the controls. Primers specific for ubiquitin were

used to normalize small differences in template amounts. For RNA gel blot

analysis, 10 mg of total RNA was denatured, fractionated by electrophoresis

on a 1% agarose gel, and then blotted onto nylon membranes (Amersham

Biosciences). Digoxigenin-labeled DNA probes were made with DIG PCR

labelingmix (BoehringerMannheim) using gene-specific forward and reverse

primer sets described earlier (Uppalapati et al., 2009). Hybridization was

performed at 658Covernight in a solution containing 50mMChurch buffer

(pH 7.2; Church and Gilbert 1984), 53 SSC (13 SSC is 0.15 M NaCl and

0.015 M sodium citrate), 0.1% laurylsarcosine, 7% SDS (w/v), and 2%

blocking solution (Boehringer Mannheim). Further washes were done at

658C in a solution containing 0.53 SSC and 0.1% SDS. Hybridized

mRNAs were detected with anti-DIG antibody conjugated with alkaline

phosphatase (Boehringer Mannheim) and its chemiluminescent sub-

strate, CDP-Star (Boehringer Mannheim).

Accession Numbers

Sequence data from this article for M. truncatula genes can be found in the

Dana Farber Cancer InstituteMedicago gene index, J. Craig Venter Institute–

Medicago Genome Resource, or GenBank/EMBL databases under the

following TC numbers or locus number: CER1-1 (TC130292), CER2

(TC115187), CER3 (TC125004), CER4-1 (TC122585), CER4-2 (TC160800),

CER6-1 (TC116151), CER6-2 (TC125487), CER6-3 (TC121408), CER6-4

(TC113573), CER8-1 (TC140235), CER10 (TC125186), PAS2 (GE348322),

KCR1 (TC113588), KCR2 (TC145787),WSD1 (TC145247), FATB (TC148248),

MYB30 (TC142663),MYB96 (Medtr4g135250/Medtr8g112130), IRG1/PALM1

(GI:298201167), and LTP (TC97568).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Expression Profiles for Selected Genes in the

Phenylpropanoid Pathway (PAL, CHS, CHR, CHI, IFS, and IFR) and

Pathogenesis-Related Genes (PR3 and PR10) in the Wild Type (R108)

at 0, 8, 24, and 48 h after Inoculation with P. emaculata Urediniospores.

Supplemental Figure 2. A Schematic Showing the Sequence of

Events for Conducting the Forward Genetic Screens to Identify M.

truncatula Genes Involved in Nonhost Resistance against P. emaculata.

Supplemental Figure 3. Expression Profiles for Selected Defense-

Related Genes and Antimicrobial Activity of Protein Extracts on

Fungal Differentiation.

Supplemental Figure 4. Rust Germ Tube Differentiation on Multiple

Mutant Alleles of irg1.

Supplemental Figure 5. Leaf Phenotype of 4-Week-Old Wild-Type

R108 and irg1-1 Plants of M. truncatula.

Supplemental Figure 6. A Simplified Wax Biosynthesis Pathway and

Some CER Genes Implicated in Wax Biosynthesis in Arabidopsis

(Modified from Kunst and Samuels, 2003; Samuels et al., 2008).

Supplemental Figure 7. Composition of Alcohols and Alkanes in

Wild-Type and irg1 Mutant Alleles of Medicago truncatula.

Supplemental Table 1. Fold Changes in Expression of Correspond-

ing M. truncatula Orthologs of Arabidopsis Wax Biosynthesis-Related

Genes Using qRT-PCR and Microarray Analysis.

Supplemental Table 2. List of Genes and Corresponding Primers

Used for qRT-PCR.

Supplemental Data Set 1. A List of Differentially Regulated Genes

(Minimum Twofold Up- or Downregulated) in Three Different Alleles of

irg1 (irg1-1/palm1-4, irg1-2 /palm1-5, and irg1-5/palm1-6).

ACKNOWEDGMENTS

We thank Markus Frank for helpful discussions, Yongfeng Zhang for

assistance with mutant screening, and Kelly Craven, Clemencia Rojas,

and Jackie Kelley for critical reading of the manuscript. We also thank

Azhaguvel Perumal and Muhammet Sakiroglu for guidance with statis-

tical analyses and Janie Gallaway and Colleen Elles for plant mainte-

nance in the greenhouse. This work was supported through a grant to

K.S.M. and S.R.U. from BASF Plant Sciences, Germany, and National

Science Foundation–Experimental Program to Stimulate Competitive

Research (EPS-0814361) to K.S.M. The insertion mutant lines of M.

truncatula were created through research support, in part, from the

National Science Foundation (DBI-0703285).

AUTHOR CONTRIBUTIONS

S.R.U. identified the mutant, designed research, analyzed data, and

wrote the article. Y.I. designed research, performed research, and

analyzed data. K.S.M. designed research and wrote the article. S.R.U.

and Y.I. performed the bulk of the experiments, and V.D. and S.M. helped

them with genetic screens. J.N. helped with scanning electron micros-

copy. M.B. performed wax analysis using GC-MS. Y.T. analyzed micro-

array data. J.C., M.T., P.R., R.C., and H.S. contributed reagents and

provided seed stocks for Tnt1 mutant populations or PALM1 comple-

mented lines.

Received October 24, 2011; revised December 16, 2011; accepted

December 31, 2011; published January 31, 2012.

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370 The Plant Cell

DOI 10.1105/tpc.111.093104; originally published online January 31, 2012; 2012;24;353-370Plant Cell

Schultheiss and Kirankumar S. MysoreJianghua Chen, Jin Nakashima, Yuhong Tang, Million Tadege, Pascal Ratet, Rujin Chen, Holger

Srinivasa Rao Uppalapati, Yasuhiro Ishiga, Vanthana Doraiswamy, Mohamed Bedair, Shipra Mittal,Reduced Spore Differentiation of Anthracnose and Nonhost Rust Pathogens

Mutants Results inMedicago truncatula irg1/palm1Loss of Abaxial Leaf Epicuticular Wax in

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