Entry Mode–Dependent Function of an Indole GlucosinolatePathway in Arabidopsis for Nonhost Resistance againstAnthracnose Pathogens W

Kei Hiruma,a,1 Mariko Onozawa-Komori,a,1 Fumika Takahashi,a Makoto Asakura,a Paweł Bednarek,b

Tetsuro Okuno,a Paul Schulze-Lefert,b and Yoshitaka Takanoa,2

a Department of Plant-Microbe Interactions, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, JapanbMax-Planck-Institut fur Pflanzenzuchtungsforschung, D-50829 Cologne, Germany

When faced with nonadapted fungal pathogens, Arabidopsis thalianamounts nonhost resistance responses, which typically

result in the termination of early pathogenesis steps. We report that nonadapted anthracnose fungi engage two alternative

entry modes during pathogenesis on leaves: turgor-mediated invasion beneath melanized appressoria, and a previously

undiscovered hyphal tip–based entry (HTE) that is independent of appressorium formation. The frequency of HTE is

positively regulated by carbohydrate nutrients and appears to be subject to constitutive inhibition by the fungal mitogen-

activated protein kinase (MAPK) cascade of MAPK ESSENTIAL FOR APPRESSORIUM FORMATION1. The same MAPK

cascade is essential for appressorium formation. Unexpectedly, the Arabidopsis indole glucosinolate pathway restricts

entry of the nonadapted anthracnose fungi only when these pathogens employ HTE. Arabidopsis mutants defective in indole

glucosinolate biosynthesis or metabolism support the initiation of postinvasion growth of nonadapted Colletotrichum

gloeosporioides and Colletotrichum orbiculare. However, genetic disruption of Colletotrichum appressorium formation

does not permit HTE on host plants. Thus, Colletotrichum appressoria play a critical role in the suppression of preinvasion

plant defenses, in addition to their previously described role in turgor-mediated plant cell invasion. We also show that HTE is

the predominant morphogenetic response of Colletotrichum at wound sites. This implies the existence of a fungal sensing

system to trigger appropriate morphogenetic responses during pathogenesis at wound sites and on intact leaf tissue.

INTRODUCTION

Nonhost resistance can be defined as immunity displayed by an

entire plant species against all genetic variants of a pathogen

species and thus affects the host range of a pathogen (Heath,

2000). Arabidopsis thaliana exhibits durable resistance against

nonadapted powdery mildew fungi such as Blumeria graminis

and Erysiphe pisi, which in nature colonize grass and pea (Pisum

sativum) species, respectively. This nonhost resistance depends

on both preinvasion and postinvasion immune responses (Lipka

et al., 2005, 2008).

PEN1, PEN2, and PEN3 of Arabidopsis were identified as

factors of preinvasion resistance against nonadapted powdery

mildewpathogens.PEN1encodes aplasmamembrane–resident

syntaxin.PEN1syntaxin formssolubleN-ethylmaleimide–sensitive

factor attachment protein receptor (SNARE) complexes together

with SNAP33 and VAMP721/VAMP722, and this SNARE com-

plex is required for focal immune responses at attempted path-

ogen entry sites in leaf epidermal cells (Collins et al., 2003; Kwon

et al., 2008). PEN2 encodes a glycosyl hydrolase that localizes to

peroxisomes (Lipka et al., 2005; Bednarek et al., 2009). Data to

this point suggest that PEN1 is engaged specifically in nonhost

resistance against powdery mildews. In contrast, PEN2 is in-

volved in resistance responses against multiple parasites, in-

cluding the nonadapted oomycete Phytophthora infestans, the

host-adapted oomycete Pythium irregulare, and the broad host

range fungal pathogen Plectosphaerella cucumerina, indicating

that metabolite(s) synthesized by PEN2 exhibit broad-spectrum

antimicrobial activity (Lipka et al., 2005). Recently, it has been

reported that PEN2 encodes an atypical myrosinase that hydro-

lyzes 4-methoxynidol-3-ylmethylglucosinolates (4MI3G) as an in

planta substrate for antifungal responses (Bednarek et al., 2009).

TheCYP81F2-encodedP450monooxygenase is essential for the

pathogen-induced accumulation of the 4MI3G PEN2 substrate

(Bednarek et al., 2009; Clay et al., 2009). Furthermore, it has been

reported that PEN2-related metabolites are required for the

activation of PMR4/GSL5-dependent callose deposition upon

treatment with flg22, a bacterial microbe-associated molecular

pattern (Clay et al., 2009). PEN3 was shown to encode an ATP

binding cassette (ABC) transporter. Genetic interaction analysis

of pen2 and pen3mutants suggested that PEN3 is likely involved

in exporting toxic compounds, including PEN2-catalyzed me-

tabolites, to sites of attempted fungal invasion (Stein et al., 2006).

The large ascomycete genusColletotrichum is one of themost

economically important groups of plant pathogens, causing

anthracnose disease on a wide range of crops (Agrios, 1988).

1 These authors contributed equally to this work.2 Address correspondence to ytakano@kais.kyoto-u.ac.jp.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: Yoshitaka Takano(ytakano@kais.kyoto-u.ac.jp).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.110.074344

The Plant Cell, Vol. 22: 2429–2443, July 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

In contrast to true obligate biotrophs such as powdery mildews,

Colletotrichum species can be cultured axenically and can be

genetically modified by stable transformation. This greatly facil-

itates mutational analysis and the critical assessment of gene

function by targeted gene disruption. Asexual spores (conidia) of

fungal pathogens such as Colletotrichum and Magnaporthe

species generally develop specialized infection structures (ap-

pressoria) that are highly pigmented with melanin, which is

critical for appressorium function (Kubo and Furusawa, 1991;

Dean, 1997). For example, the formation of a melanin layer

enables appressoria to generate the high turgor pressure nec-

essary for mechanical penetration through the plant cuticle and

cell wall (Howard and Valent, 1996; de Jong et al., 1997;

Bechinger et al., 1999). Consistent with this, melanin biosynthe-

sis inhibitors block the functionality of appressoria of both

Colletotrichum and Magnaporthe species. It is also known that

many Colletotrichum species and Magnaporthe oryzae use a

hemibiotrophic infection strategy during postinvasion growth, in

which these pathogens initially grow biotrophically in living host

cells before switching to a destructive necrotrophic phase of

infection (Perfect et al., 1999; Koga et al., 2004).

Isolates of Colletotrichum higginsianum infect Arabidopsis

(O’Connell et al., 2004; Narusaka et al., 2004), which provides a

new model pathosystem between Arabidopsis and Colletotri-

chum in which both partners in the interaction can be genetically

manipulated. We previously reported that nonhost resistance of

Arabidopsis against Colletotrichum species largely depends on

preinvasion immune responses that act at the cell periphery

(Shimada et al., 2006). It was shown that PEN1 syntaxin is

dispensable for preinvasion nonhost resistance to Colletotri-

chum. Furthermore, focal accumulation of PEN1 toward entry

sites of nonadapted Colletotrichum species was not detected

(Shimada et al., 2006). This demonstrated that molecular com-

ponents of preinvasion immune responses differ between differ-

ent ascomycete fungal parasites, even though these employ the

same infection strategy of direct entry into host epidermal cells

mediated by an appressorium. Recently, PEN2 was found to be

involved in basal preinvasion resistance of Arabidopsis against

the host-adapted pathogen C. higginsianum (Huser et al., 2009).

However, it remained unknown whether the PEN2-dependent

indole glucosinolatemetabolism pathway contributes to nonhost

resistance against Colletotrichum species.

In this study, we show that nonadapted Colletotrichum gloeo-

sporioides, a mulberry (Morus nigra) anthracnose fungus, in-

duces slight lesions on pen2 mutants but not on wild-type

Arabidopsis plants, thereby demonstrating the involvement of

PEN2 in nonhost resistance to this pathogen. We found that

inoculation with this nonadapted fungus in the presence of

monosaccharides such as Glc enhanced lesion development

on Arabidopsis mutants lacking PEN2 myrosinase or other

enzymes required for the biosynthesis of its substrate, 4MI3G.

Glc markedly inhibited the development of melanized appres-

soria. Surprisingly, in the nonhost interaction between C. gloeo-

sporioides (hereafter called Cg) and Arabidopsis pen2 mutants,

we found that the pathogen develops intracellular hyphae from

undifferentiated hyphal tips but not beneath normal melanized

appressoria. Further analyses of a range of Colletotrichum mu-

tants revealed the existence of an alternative hyphal tip–based

entry (HTE) mode independent from the formation of melanized

appressoria. We infer a model in which the fungal mitogen-

activated protein kinase (MAPK) cascade of MAPK ESSENTIAL

FOR APPRESSORIUM FORMATION1 (MAF1) exerts an inhibi-

tory function on HTE, which in turn can be derepressed by a

favorable nutritional status. In addition, we show that HTE is the

predominant morphogenetic response of Colletotrichum during

pathogenesis at wound sites.

RESULTS

PEN2 Contributes to Nonhost Resistance against Cg

To assess the potential involvement of PEN2 in nonhost resis-

tance to Colletotrichum, two independent pen2mutants (pen2-1

and pen2-2) were inoculated with conidiospores of the non-

adapted speciesColletotrichum orbiculare (isolate 104-T), which

infects cucumber (Cucumis sativus), and Cg (isolate S9275),

which infects mulberry. No macroscopic disease symptoms

were observed on pen2, pen1, and Columbia (Col-0) wild-type

plants upon inoculation with C. orbiculare conidiospores (data

not shown). However,Cg induced slight leaf lesions on bothpen2

mutants, but not on pen1 or wild-type plants (Figure 1A, top

panel), suggesting the involvement of PEN2 in nonhost resis-

tance against Cg. At 3 to 4 d post inoculation (dpi) on Arabidop-

sis, the host-adapted C. higginsianum fungus forms necrotic leaf

lesions that eventually consume large parts of leaf tissue at a

distance from droplet inoculation sites (Figure 1A, bottom panel).

This differs from the lesions caused byCgdroplet inoculations on

pen2 leaves, which became noticeable as small white flecks at 3

to 4 dpi and hardly expanded even at 7 dpi.

Microscopic inspection of inoculated leaf tissue revealed that

the host-adapted C. higginsianum effectively developed intra-

cellular biotrophic hyphae that are readily stained by trypan blue

at 60 h post inoculation (hpi; see Supplemental Figure 1A online).

In the nonhost interaction between pen2 plants and Cg, these

intracellular hyphae were detected only occasionally (see Sup-

plemental Figure 1B online). At 60 hpi,Cg cellular entry invariably

resulted in the death of plant cells that contained intracellular

hyphae and often resulted in the death of their adjacent cells, as

indicated by trypan blue retention. This contrasts with rarely

detectable trypan blue staining of host cells in interactions with

the host-adapted C. higginsianum during this early biotrophic

infection phase. Together, these findings point to the activation

of postinvasion defense responses against nonadapted Cg

sporelings on pen2 plants.

Cg Induces Enhanced Leaf Lesion Formation in the

Presence of Glc

Notably, when Cg conidia were inoculated as droplet suspen-

sions in 0.1% Glc on pen2 plants, lesion formation was dramat-

ically enhanced in comparison with inoculations without Glc

(Figures 1B and 1C), although the addition of nutrients was

previously reported to inhibit appressorium development of

the avocado (Persea americana) isolate of Cg (Hwang and

Kolattukudy, 1995). On the other hand, Glc-supplemented

2430 The Plant Cell

Figure 1. PEN2 Is Involved in Resistance against Nonadapted Cg.

(A) The nonadapted Cg formed slight lesions on the pen2 mutants, whereas it did not form any lesions on wild-type (Col-0) plants. The adapted C.

higginsianum (Ch) formed clear lesions on Col-0 at 4 dpi, and the lesions were large enough to cause severe collapse at 7 dpi. In contrast, lesions

induced by Cg did not extend at 7 dpi. The experiment was repeated three times with similar results.

(B) Inoculation ofCgwith Glc results in enhanced lesion formation on the pen2mutants. Conidial suspensions ofCg, with 0.1%Glc (+ glu) or without Glc

(� glu), were drop-inoculated on the tested plants and incubated for 4 d.

(C) Quantitative analysis of lesion development by Cg on Arabidopsis. Conidial suspensions of Cg were spot-inoculated on the tested plants with 0.1%

Glc (+ glu) or without Glc (� glu). Lesion development at spotted sites (n = 50 per plant) was assessed at 4 dpi and assigned to three types. 1, no lesion;

2, slight lesion; 3, severe lesion. The means and SD were calculated from three independent experiments.

(D) Glc partially inhibits the formation of melanized Cg appressoria. When inoculated on Col-0 plants in the absence of Glc, most conidia developed

well-melanized appressoria (class 1). In the presence of Glc, conidia failed to form well-melanized mature appressoria effectively but instead developed

appressoria with slight melanization (class 2) or appressoria without detectable pigmentation (class 2). Conidia also formed tiny appressoria without

melanization (class 3) or did not form swollen structures (class 3). At least 100 conidia were inspected in each experiment. The means and SD were

calculated from three independent experiments.

(E) Infection-related development ofCg categorized to class 1 to class 3. Conidial suspensions of Cgwere inoculated on Arabidopsiswild type with Glc,

and inoculated plants were incubated for 14 h. C1, Class 1; C2, class 2; C3, class 3. Bars = 10 mm.

Fungal Entry Mode and Nonhost Resistance 2431

conidiospore inoculation did not result in lesion formation

on wild-type and pen1 plants (Figures 1B and 1C). Thus, Glc

appeared to enhance specifically the virulence of Cg on pen2

plants. Lesion formation by Cg was also enhanced by the

addition of Suc but not sorbitol (see Supplemental Figure 2A

online).

In the presence of Glc, Cg germinated effectively, but there-

after the differentiation of the fungal sporelings was impaired

such that the incidence of melanized appressoria was greatly

reduced (Figures 1D and 1E). To better characterize the Glc-

mediated changes to Cg infection structures on the leaf surface,

we assigned individual germinated spores to three morphotypes

(Figures 1D and 1E). Class 1 sporelings differentiated darkly

melanized appressoria and are indistinguishable from the prev-

alent surface infection structure of the fungus in the absence of

Glc. Class 2 sporelings differentiated an appressoriumwith slight

pigmentation or an appressorium without detectable pigmenta-

tion. Finally, class 3 sporelings produced a long germ tube

without a recognizable appressorium or differentiated a tiny

swollen structure at the hyphal tip. In the absence of Glc,;70%

of germinated conidia formed melanized mature appressoria

(class 1) on Arabidopsis leaves. In the presence of Glc, the

incidence of class 1 sporelings decreased, whereas the inci-

dence of class 3 morphotypes dramatically increased (from

;3% to;26%; Figure 1D). Suc also interfered with the regular

development of melanized appressoria, but sorbitol had little

effect (see Supplemental Figure 2B online). In contrast toCg, the

host-adapted C. higginsianum developed typical melanized ap-

pressoria on Arabidopsis, and lesion formation was not en-

hanced in the presence of 0.1% Glc (see Supplemental Figure 3

online).

Cg Entry into pen2 Plants Occurs Independently of

AppressoriumMelanization

To investigateCg entry in the presence ofGlc in greater detail, we

expressed green fluorescent protein (GFP) in a transgenic Cg

strain to visualize infection structures by confocal laser micros-

copy. This revealed invasive growth of Cg sporelings on pen2

plants at ;35% of interaction sites, whereas fungal entry was

essentially undetectable on wild-type and pen1 plants (Figure

2A). We noticed that on pen2 plants, most sporelings with darkly

melanized appressoria (class 1) failed to form intracellular hy-

phae (Figures 2B and 2C). Remarkably, however,;40%of class

2 and class 3 sporelings had effectively initiated invasive fungal

growth (Figures 2B and 2C). These results suggest that Cg

invasion on pen2 plants occurs independently of appressorium

melanization and that Glc-mediated inhibition of appressorium

development results in enhanced fungal entry.

The findings described above are unexpected because it is

generally accepted that defects in the formation of melanized

appressoria of both Colletotrichum and Magnaporthe species

result in loss or decrease of pathogenicity on host leaves (Kubo

and Furusawa, 1991; Howard and Valent, 1996). Melanized

appressoria generate turgor pressure, which is thought to pro-

vide the mechanical force to penetrate plant cell walls (Howard

and Valent, 1996; de Jong et al., 1997; Bechinger et al., 1999).

Therefore, effective invasion on pen2 plants without melanized

appressoria appears to contradict previous findings on turgor

pressure–driven fungal entry of this filamentous pathogen.

We used the melanin synthesis inhibitor carpropamid (Hattori

et al., 1994) to explore further the existence of an alternative

Colletotrichum cellular entrymode.We first investigated whether

appressorium melanization is critical in the interaction between

Arabidopsis and the host-adapted C. higginsianum. When co-

nidial suspensions of C. higginsianum were inoculated together

with carpropamid on Arabidopsis Col-0, germinated conidia

formed nonmelanized appressoria (Figure 3A) that failed to

induce leaf lesions (Figure 3B). This is consistent with a require-

ment of appressorium melanization for pathogenicity of C.

higginsianum. When Cg conidial suspensions containing car-

propamid alone or with Glc were inoculated on leaves, the

sporelings failed to develop melanized appressoria but retained

the ability to induce leaf lesions on pen2 plants (Figure 3B; see

Supplemental Figure 4 online). These results suggest that Cg

caused lesions on pen2 plants via an alternative cellular entry

route for which melanization of appressoria is dispensable.

We have previously shown that in nonhost interactions of

Arabidopsis and Colletotrichum, the initiation of penetration peg

formation, a pathogenesis step seen subsequent to appresso-

rium melanization, is necessary to induce the formation of de

novo synthesized host cell wall material (Shimada et al., 2006).

Such cell wall appositions (also called papillae) are deposited

onto the existing plant cell wall in the paramural space directly

underneath fungal penetration pegs and are a common early

defense response to filamentous pathogens that must penetrate

the host cell wall during pathogenesis (Meyer et al., 2009). We

tested for the presence of papillary callose, amajor constituent of

pathogen-induced papillae, at Cg entry sites on pen2 plants by

aniline blue staining (Jacobs et al., 2003). Both class 2 and class

3 Cg sporelings were found to induce the accumulation of

papillary callose beneath attempted and successful fungal entry

sites (Figure 3C). This provides indirect evidence that Cg forms

penetration peg–like structures during the melanization-inde-

pendent entry into plant cells. Consistent with this interpretation,

callose deposition was excluded from the center of papillae,

where a penetration peg is normally situated (Figure 3C).

The PEN2 Indole Glucosinolate Pathway Restricts Cg Entry

PEN2 myrosinase is known to hydrolyze 4MI3G as an in planta

substrate, which in turn originates from unsubstituted indol-3-

ylmethylglucosinolate followed by a conversion mediated by the

P450 monooxygenase CYP81F2 (Bednarek et al., 2009; Pfalz

et al., 2009). A precursor of indol-3-ylmethylglucosinolate is

generated by the P450monooxygenase-catalyzed conversion of

Trp to indole-3-acetaldoxime and is mediated by the genetically

redundant CYP79B2 and CYP79B3 gene products (Zhao et al.,

2002). This indole glucosinolate biosynthesis and metabolism

pathway is essential to restrict the entry of nonadapted powdery

mildew fungi (Bednarek et al., 2009). To find out whether the

same indole glucosinolate biosynthesis/metabolism pathway is

required to blockCg entry, we evaluated infection phenotypes on

Arabidopsis cyp79B2 cyp79B3 double and cyp81F2 single mu-

tants. Glc-supplemented Cg conidiospore suspensions induced

leaf lesion formation, essentially phenocopying the infection type

2432 The Plant Cell

seen on pen2 plants (Figure 4A). These results suggest that the

biosynthesis of 4MI3G and its hydrolysis by PEN2 myrosinase

are necessary for defense responses toCg prior to and/or during

entry into plant cells (preinvasion defense). In contrast, the pad3

mutant, in which the Trp-derived biosynthesis of the antimicro-

bial camalexin is blocked (Zhou et al., 1999), showed an infection

phenotype that is indistinguishable from the wild type, suggest-

ing that this phytoalexin does not contribute to preinvasion

defense responses against Cg (Figure 4A).

Powdery mildew–induced callose synthesis at incipient entry

sites largely depends on the glucan synthase–like protein PMR4/

GSL5 (Jacobs et al., 2003; Nishimura et al., 2003). PMR4/GSL5-

dependent callose accumulation is also induced by the FLS2

receptor kinase upon detection of the bacterial microbe-associ-

ated molecular pattern flg22, and this appears to engage a

signaling pathway involving CYP81F2, PEN2, as well as the

plasma membrane-resident ABC transporter PEN3 (Clay et al.,

2009). To assess whether PMR4/GSL5 callose contributes to

PEN2-dependent preinvasion resistance against Cg, pmr4

plants were inoculated with Cg spore suspensions supple-

mented with Glc. Unlike the Cg-induced leaf lesions on pen2,

cyp79B2 cyp79B3, and cyp81F2 plants, pmr4 plants exhibited

an infection phenotype that was indistinguishable from the wild

type (Figure 4A). However, Cg induced leaf lesions on pen3

plants, although their severity appeared to be reduced compared

with those onpen2, cyp79B2 cyp79B3, or cyp81F2plants (Figure

4A). We thus determined the entry rate ofCg on pen3 plants, and

it was indeed intermediate (8.3%6 2.4%) between that of pen2

plants (30.6% 6 6.6%) and that of the almost fully resistant wild

type (0.4% 6 0.4%; Figure 4B). This supports a positive rela-

tionship between Cg entry rates and lesion severity. Since pen3

plants were shown to hyperactivate the salicylic acid (SA) sig-

naling pathway following pathogen attack, resulting in enhanced

disease resistance against a host-adapted powdery mildew

(Stein et al., 2006), we also investigated pen3 sid2 double

mutants, in which pathogen-induced SA accumulation is abol-

ished due to the absence of isochorismate synthase in the SA

biosynthesis pathway (Wildermuth et al., 2001). Entry rates in

pen3 and pen3 sid2 plants were indistinguishable upon Cg

challenge (Figure 4B), indicating that potentially hyperactivated

SA signaling in pen3 plants does not confound the infection

phenotype. We thus conclude that, unlike in interactions with

nonadapted powdery mildews, the bulk of PEN2-derived me-

tabolites acts independently of the PEN3 ABC transporter to

terminate Cg entry attempts.

We next tested by quantitative RT-PCR analysis whether gene

expression of the PEN2-dependent indole glucosinolate path-

way is responsive to Cg challenge on Col-0 wild-type plants.

Expression of PEN2, PEN3, CYP79B2, and CYP81F2 was each

found to be pathogen inducible as early as 9 hpi (Figure 5A).

Because Cg fails to invade plant cells of wild-type Arabidopsis,

Figure 2. Invasion of the pen2 Mutants by Cg Uncoupled with Devel-

opment of Mature Melanized Appressorium.

(A) Ratio of inoculated Cg conidia that successfully invaded plant cells.

Conidia ofCg expressing GFP were inoculated on the tested Arabidopsis

lines in the presence of Glc. Inoculated plants were incubated for 14 h.

Invasion of germinating conidia was investigated by confocal micros-

copy. At least 100 conidia were observed in each experiment. The means

and SD were calculated from three independent experiments.

(B) Formation of invasion hyphae by aberrant fungal structures. Conidia

of Cg expressing GFP were inoculated on the pen2 mutant, and inoc-

ulated plants were incubated for 14 h. Top images show invasion via

nonmelanized appressorium (class 2 [C2]) into the pen2 mutant. Bottom

images show invasion via a tiny swollen structure (class 3 [C3]), whereas

the melanized appressorium (class 1 [C1]) failed to develop an intracel-

lular hypha (ih). Left images are focused on fungal structures formed on

the plant surface, whereas right images are focused on intracellular

hypha. In each pair of images, the left is the light micrograph of the same

cells shown via confocal microscopy on the right. Bars = 10 mm.

(C)Quantitative data on invasion manner of Cg into the pen2mutant. The

percentage of invasion by each class structure (classes 1–3) was based

on the total number of each class structure. The means and SD were

calculated from three independent experiments.

Fungal Entry Mode and Nonhost Resistance 2433

these expression data suggest that the nonhost plant detects the

attack of Cg during the preinvasion pathogenesis phase to

stimulate the coordinated expression of an inducible preinvasion

defense pathway.

PEN2 myrosinase has been shown to localize to peroxisomes

(Lipka et al., 2005). We investigated the dynamics of PEN2-

containing peroxisomes in the interaction with Cg by using a

transgenic pen2-1 line expressing functional PEN2-GFP. In

healthy plants, mobile PEN2-GFP–marked compartments are

randomly distributed within single leaf epidermal cells (Lipka

et al., 2005). At 16 h after inoculation with Cg conidial suspen-

sions supplemented with Glc, PEN2-GFP signals were prefer-

entially found to be concentrated underneath attempted entry

sites of class 2 (39% of class 2 sites had associated PEN2-GFP

signal; n = 100) and class 3 sporelings (35%) but occasionally

also beneath class 1 sporelings (3%; Figure 5B). This is consis-

tent with the idea that presumed PEN2-derived antimicrobials

are produced at high concentrations at incipient fungal entry

sites.

Host-Adapted and Nonadapted Colletotrichum Establish

Intracellular Hyphae by Host Plasma

Membrane Invagination

Intracellular hyphae of the host-adapted pathogen C. higginsia-

num are surrounded by the invaginated host plasma membrane,

and this accommodation of the infection structure appears to be

critical for the establishment of a transient biotrophic lifestyle of

the fungus (Shimada et al., 2006; Figure 6A). We investigated

whether the melanization-independent Cg entry mode in Arabi-

dopsis cells results in a similar accommodation of intracellular

hyphae through plasma membrane invagination. To better visu-

alize the fungal invasion process, we generated a transgenic

Arabidopsis line expressing functional PEN1-GFP in a pen2

background in which the fluorescent syntaxin fusion protein

marks the plant plasma membrane (Collins et al., 2003; Pajonk

et al., 2008). To distinguish the plant plasma membrane from

intracellular fungal hyphae, we generated a transgenic Cg strain

expressing a red fluorescent protein (RFP) and inoculated con-

idiospores together with Glc on pen2-1 plants expressing PEN1-

GFP. Confocal microscopic imaging revealed that intracellular

hyphae of Cg were indeed surrounded by PEN1-GFP fluores-

cence on pen2 plants (Figure 6B). A similar although much

reduced PEN1-GFP fluorescence signal surrounding intracellu-

lar hyphae was detected in the PEN2 background upon chal-

lenge with the host-adapted C. higginsianum (Figure 6A). This

suggests that both host-adapted and nonadapted Colletotri-

chum species establish intracellular hyphae through invagination

of the host plasma membrane. The much stronger PEN1-GFP ac-

cumulation at intracellular hyphae in interactions with Cg could

Figure 3. Cg Has an Alternative Entry Mode That Is Resistant to a

Melanin Biosynthesis Inhibitor.

(A)Conidial suspensions of the adaptedC. higginsianumwere inoculated

on Arabidopsis Col-0 in the presence of carpropamid. At 3 dpi, inocu-

lated leaves were subjected to trypan blue staining. C. higginsianum

formed melanized appressoria (ap) in the absence of carpropamid, and

melanized appressoria developed intracellular hyphae (ih) stained with

the dye (left panel). In the presence of carpropamid, they formed

nonmelanized appressoria without formation of intracellular hyphae

(right panel). Bars = 10 mm.

(B) Effects of carpropamid on lesion development. Addition of carpro-

pamid results in failure of lesion formation following inoculation of wild-

type Col-0 with the adapted C. higginsianum (Ch). On the other hand,

after the inoculation of the nonadapted Cg in the presence of Glc, the

fungus forms lesions on the pen2 mutants even in the presence of

carpropamid. The photograph was taken at 4 dpi. This experiment was

repeated three times with similar results.

(C) Papilla formation at the invasion trial site of Cg via aberrant fungal

structures. Conidial suspension of Cg was inoculated on the pen2-1

mutant. Inoculated plants were incubated for 16 h and subjected to aniline

blue staining to detect papillary callose (right images); light micrographs of

the same regions are shown on the left. Top photographs, An appresso-

rium with slight melanization (class 2) induced papillary callose accumu-

lation. Bottom photographs, Elongated germ tube (class 3) induced

papillary callose accumulation. Arrows indicate the site of penetration

peg formation. C1, Class 1; C2, class 2; C3, class 3. Bars = 20 mm.

2434 The Plant Cell

point to enhanced postinvasion secretory activity at the interface

of the fungal hypha that might be suppressed by the host-

adapted fungus. Plasmolysis experiments also suggested that

epidermal cells of pen2 plants, containing intracellular Cg hy-

phae, remain alive during the early stage of invasive growth (see

Supplemental Figure 5 online). Taken together, these findings

support the conclusion thatCg can at least transiently establish a

biotrophic phase of infection in the absence of indole glucosi-

nolate–dependent preinvasion defense responses.

To find out whether the appressorium-independent entry

mode ofCg into Arabidopsis cells is conserved on other nonhost

plants, we inspected the colonization of Cg on cucumber, which

is distantly related to Arabidopsis.Cg formed melanized appres-

soria on cucumber cotyledons in the absence of Glc, while the

presence of Glc inhibited the formation of melanized appressoria

(Figure 7A). Unlike Cg sporelings on Arabidopsis pen2 leaves,

however, Cg failed to form lesions on cucumber in the presence

of Glc (Figure 7B), presumably due to preinvasion defense

responses. Since it was previously shown that heat shock

treatment of cucumber cotyledons impairs inducible defense

responses (Takano et al., 2006), we also examined Cg spore

droplet inoculations in the presence of Glc after a 30-s exposure

of cucumber cotyledons to heat stress. Under these conditions,

Cg conidiospore inoculation resulted in severe lesion formation

on cotyledons (Figure 7B), which, at the microscopic level, was

associated with the development of invasion hyphae (Figure 7C).

Together, these data indicate that the appressorium-indepen-

dent Cg entry mode can be executed on two divergent nonhost

plants when their defense is blocked either genetically or by

physical stress. However, when Cgwas inoculated on its natural

host, mulberry, without Glc, the fungus formed melanized ap-

pressoria that subsequently developed invasion hyphae. This

indicates that Cg utilizes different pathogenesis routes for cel-

lular entry on host and nonhost plants (see Supplemental Figure

6 online).

Genetic Uncoupling of Appressorium Formation from

Cellular Entry

To find out whether Colletotrichum species other than Cg can

also enter leaf epidermal cells in the absence of melanized

appressoria and whether fungal entry can be genetically uncou-

pled from appressorium development, we used theC. orbiculare

maf1mutant strain. C. orbiculare MAF1 encodes a MAPK that is

essential for the early differentiation phase of appressorium

formation, and the maf1 strain forms elongated germ tubes

without appressoria on both host plants and glass (Kojima et al.,

2002). On the nonhost Arabidopsis, the maf1 strain germinated

effectively and formed elongated hyphae-like structures without

appressoria, while thewild-type strain ofC. orbiculare developed

normal melanized appressoria (Figure 8A). Droplet inoculation of

themaf1 fungal strain in the absence of Glc on Arabidopsis wild-

type or pen1 plants did not result in leaf lesion formation, while

inoculations on pen2 plants gave rise to marked lesions (Figure

8B). Importantly, wild-typeC. orbiculare failed to produce lesions

on any of these plant genotypes (Figure 8B), thus demonstrating

that lesion formation occurs only when appressorium develop-

ment in the fungus is blocked and Arabidopsis cannot generate

PEN2-derived metabolites. This finding also excludes the pos-

sibility that the presence of Glc/Suc in Cg conidial suspensions

indirectly facilitates entry of the nonadapted fungus on pen2

plants.

To validate that lesion formation induced by the C. orbiculare

maf1 strain on pen2 plants is associated with the formation of

intracellular hyphae similar to those seen upon inoculation with

Cg in the presence of Glc, we microscopically inspected pen2

plants expressing PEN1-GFP. This revealed that the maf1 strain

forms intracellular hyphae surrounded by the host plasma mem-

brane marker PEN1-GFP in the pen2 background (Figure 8C).

Taken together, these results demonstrate that at least two

nonadapted Colletotrichum species can enter Arabidopsis pen2

leaf epidermal cells independently of appressorium develop-

ment.

We inoculated pen2 plants with two additional nonpatho-

genic C. orbiculare strains, lacking SCD1 orMTK1 (Kubo et al.,

1996; Tsuji et al., 2003; see Supplemental Figures 7–9 online).

While the former gene encodes a scytalone dehydratase that is

essential for melanin biosynthesis and pathogenicity, the gene

product of MTK1 exhibits high sequence relatedness to MAPK

Figure 4. Requirement for PEN2-Activated Metabolite in Nonhost De-

fense against Cg.

(A) Conidia of Cg were inoculated in the presence of Glc on Arabidopsis

mutants defective in the PEN2 pathway (pen2-1, cyp79B2 cyp79B3,

cyp81F2, and pen3-1), the pad3 mutant, and the pmr4 mutant. Inocu-

lated plants were incubated for 4 d.

(B) Entry control of the pen3 mutant against Cg. Conidia of Cg express-

ing GFP were inoculated in the presence of Glc on the tested plants.

Inoculated plants were incubated for 14 h and subjected to analysis by

fluorescence microscopy to investigate fungal invasion. At least 100

conidia were observed in each experiment. The means and SD were

calculated from two independent experiments.

Fungal Entry Mode and Nonhost Resistance 2435

kinase kinase (MAPKKK) andwas thus designatedC. orbiculare

MAP triple kinase 1 (see Supplemental Figure 7 online). We

generated the mtk1 mutant by targeted gene disruption. The

mtk1 mutant had reduced pathogenicity on cucumber (see

Supplemental Figure 8 online) and phenocopied the maf1

mutant on a glass surface (see Supplemental Figure 9 online),

suggesting that Mtk1 is a potential MAPKKK that functions in

theMaf1MAPK pathway. In contrast, the scd1 strain retains the

ability to differentiate appressoria but exhibits a defect in

appressorium melanization and lacks pathogenicity on cucum-

ber (see Supplemental Figure 8 online). Of note, whereas the

scd1 strain failed to form lesions on pen2 plants, themtk1 strain

induced leaf lesions similar to the maf1 mutant (see Supple-

mental Figure 9 online). These data strongly suggest that the

Maf1 pathway, rather than melanin biosynthesis, negatively

regulates the utilization of an alternative C. orbiculare entry

pathway into plant cells that is independent of appressorium

development.

Figure 5. PEN2 Recruitment toward Sites of Attempted Invasion by Aberrant Fungal Structures.

(A) Induced gene expression of PEN2, PEN3, CYP79B2, and CYP81F2 by inoculation of Cg in the presence of Glc. Cgwas inoculated with 0.1% Glc on

wild-type Col-0 plants. Gene expression of PEN2, PEN3, CYP79B2, and CYP81F2 was investigated at 9 hpi by quantitative RT-PCR (I). As a control, a

0.1% Glc solution was sprayed on the tested plants, and gene expression was investigated 9 h later (NI). The means and SD were calculated from three

independent experiments.

(B) Focal accumulation of PEN2-GFP at sites of Cg invasion attempts. Cg expressing RFP was inoculated on transgenic pen2-1 plants expressing

functional PEN2-GFP fusion protein. Inoculated plants were incubated for 16 h and subjected to analysis by fluorescence microscopy. Topmicrographs

(light image on the left and merged image of GFP and RFP on the right), PEN2-GFP preferentially localizes under appressoria with slight melanization

categorized to class 2 (C2). Also, PEN2 was recruited under a melanized appressorium categorized to class 1 (C1). Bottom images, PEN2 was recruited

under a tiny swollen structure categorized to class 3 (C3). Focal accumulation sites of PEN2-GFP are indicated by arrows. Bars = 10 mm.

2436 The Plant Cell

HTE Is the Major Morphogenetic Pathogenesis Route at

Wound Sites

Next, we tested whether HTE is physiologically relevant (i.e.,

whether HTE can become the dominant mode of Cg entry

without exogenously applied Glc). It is assumed that wound

sites on plant tissue release carbohydrate nutrients, including

Glc. We tested whether Cg sporelings utilize HTE at wound sites

on Arabidopsis leaves. Cg was inoculated without Glc on leaves

that were wounded by a toothpick. Intriguingly, Cg did not form

melanized appressoria at the wound margins of wild-type and

pen2 plants but formed small colorless appressoria or elongated

hypha-like structures, which is reminiscent of the droplet inoc-

ulation of Cg spores on intact leaves in the presence of Glc

(Figure 9A). Abrading the leaves with carborundum also mark-

edly reduced the frequency of appressorium development,

suggesting that the wound-associated fungal morphogenesis

is triggered independently of different modes of wounding.

Importantly, Cg effectively formed intracellular hyphae at the

wound margins of pen2 plants but not of wild-type plants.

Intracellular hyphae at wound margins were surrounded by the

plant plasma membrane labeled by PEN1-GFP, indicating the

successful completion of HTE (Figure 9C). This points to a critical

role of indole glucosinolates in restricting Cg entry at wound

sites, where HTE is the predominant type of fungal pathogenesis.

Furthermore, we found that the nonadapted C. orbiculare also

switches to HTE at wound margins of Arabidopsis leaves. The

wild-type strain of C. orbiculare represses the formation of a

melanized appressorium at wound margins, which is similar to

the maf1 mutant phenotype (Figure 9B). At wound margins on

pen2 plants, but not onwild-type plants, the wild-type strain ofC.

orbiculare formed intracellular hyphae (Figure 9C). Taken to-

gether, these data suggest that both nonadapted anthracnose

fungi can distinguish damaged from intact leaf tissue to initiate

appropriate morphogenetic responses during preinvasion path-

ogenesis. This morphogenetic switch appears to be phyloge-

netically conserved, at least in C. orbiculare and Cg.

Figure 6. Plant Membrane Invagination in Response to Fungal Invasion

Uncoupled with Melanized Appressorium.

(A) Membrane invagination in host interaction of Arabidopsis with the

adapted C. higginsianum (Ch). C. higginsianum expressing RFP was

inoculated on Arabidopsis expressing PEN1-GFP. Inoculated plants

were incubated for 48 h. The left micrograph is focused on an appres-

sorium (indicated by a dotted circle) formed on the plant surface,

whereas the right micrograph is focused on an intracellular hypha (ih)

labeled by RFP. Bar = 10 mm.

(B) Membrane invagination in Arabidopsis pen2 mutant against invasion

via aberrant fungal structure of the nonadapted Cg. Cg expressing RFP

was inoculated with Glc on the Arabidopsis pen2 mutant expressing

PEN1-GFP. Inoculated plants were incubated for 14 h. Bar = 10 mm.

Figure 7. Infection Behavior of Cg on a Nonhost Cucumber.

(A) Cg does not form melanized appressoria on a nonhost cucumber

cotyledon in the presence of Glc. Conidial suspensions of Cg were

inoculated on the lower surface of cucumber cotyledons with (+glu) or

without (�glu) Glc and incubated for 14 h. Bars = 20 mm.

(B) Cg formed lesions on cucumber cotyledons treated with heat stress.

Conidial suspensions of Cg were inoculated onto heat-shocked (H.S.)

cotyledons of cucumber. The inoculated plants were incubated for 7 d.

(C) Formation of invasion hyphae by aberrant fungal structures. Conidia

of Cg were inoculated on heat-shocked cucumber cotyledons, and

inoculated plants were incubated for 16 h. The top micrograph focuses

on fungal structures formed on the plant surface, whereas the bottom

micrograph focuses on intracellular hypha (ih). Bars = 20 mm.

Fungal Entry Mode and Nonhost Resistance 2437

DISCUSSION

Here, we have described a pathogen entry route–dependent

function of a plant secondary metabolite pathway for extracel-

lular defense against two nonadapted Colletotrichum species.

Arabidopsis PEN2, redundantly acting CYP79B2 CYP79B3, and

CYP81F2 each limits cell entry of nonadapted Colletotrichum

germlings only when the fungus utilizes an otherwise cryptic

entrymode that bypasses the default developmental program for

appressorium-mediated invasion of the leaf epidermis. This

alternative entry mode is preferentially used by the pathogen in

the presence of environmental cues such as Glc or Suc (Figure

2C) or upon genetic disruption of appressorium formation (Fig-

ure 8).

Why is the Arabidopsis indole glucosinolate biosynthesis and

PEN2-dependent metabolism pathway dispensable for nonhost

resistance during regular Colletotrichum pathogenesis on leaves

(i.e., upon attempted entry by melanized appressoria)? The

presence of papillary callose with a penetration hole below

Colletotrichum entry sites lacking appressoria provided indirect

evidence for the differentiation of penetration peg-like structures

(Figure 3C). This is indicative of a marked constriction of the

hypha for the initiation of invasion. This and the light microscopic

inspection of Cg sporelings on leaves indicate a HTE of conidial

germ tubes without appressoria, a feature shared with Colleto-

trichum entry following differentiation of melanized appressoria.

Thus, within the limits of light microscopic inspection and with

respect to papillary callose formation as a response marker to

microbial attack, both Colletotrichum invasion processes are,

per se, not recognizably different. Consistent with this, intracel-

lular hyphae are formed by invagination of the plant plasma

membrane irrespective of the presence or absence of appres-

soria (Figures 6 and 8). However, melanized appressoria of the

host-adapted C. higginsianum started to differentiate visible

intracellular hyphae at;48 hpi, while Cg formed these infection

structures on pen2 plants much earlier, at;14 hpi. Of note, Cg-

stimulated PEN2, PEN3, CYP79B2, and CYP81F2 expression

was detectable even earlier, at 9 hpi (Figure 5A), indicating that

Arabidopsis perceives the attack on the leaf surface and mounts

defense responses to the intruder prior to the initiation of invasive

growth. This is consistent with previous work on nonpathogenic

Colletotrichum lindemuthianum mutant strains, showing that on

leaves of its host, Phaseolus vulgaris, the formation of nonfunc-

tional appressoria (i.e., failure to grow inside plant cells) was

sufficient to induce plant defense responses (oxidative burst and

PR protein accumulation; Veneault-Fourrey et al., 2005).

Since the PEN2-dependent indole glucosinolate metabolism

pathway represents an early inducible preinvasion defense path-

way (Bednarek et al., 2009;Clay et al., 2009) and is rate limiting for

Cg entry at ;14 h after inoculation, its activity underneath Cg

appressoria might be masked by additional late-acting and

redundant defense pathways. In this scenario, preinvasion de-

fense to nonadaptedCg andC. orbicularebecomes vulnerable to

Figure 8. Inoculation Assays using the C. orbiculare maf1 Mutant

Defective in Appressorium Formation.

(A) Morphology of the wild-type strain (WT; left panel) and the maf1

mutant (maf1; right panel) ofC. orbiculare (Co) on Arabidopsis. Conidia of

the tested strains were inoculated on Arabidopsis, and the inoculated

plants were incubated for 12 h. Bars = 10 mm.

(B) Themaf1mutant formed lesions on pen2mutants. Inoculation with C.

orbiculare wild type did not induce lesions on the tested plants. On the

other hand, inoculation with the maf1 mutant caused lesions on pen2

mutants but not on Col-0 or the pen1mutant. The photograph was taken

at 4 dpi. This experiment was repeated four times with similar results.

(C) Intracellular hypha of the maf1 mutant was encased by plasma

membrane of the Arabidopsis pen2 mutant. Conidia of the maf1 mutant

expressing RFP were inoculated on the pen2 mutant expressing PEN1-

GFP. Top micrograph, Merged images of XY series of RFP fluorescence

representing fungal conidium, elongated germ tube, and intracellular

hypha. Bottom micrograph, Single optical XY image of RFP and GFP

fluorescence. The dotted line indicates conidia and elongated germ tube

of the maf1 mutant. The micrograph was taken at 24 hpi. Bars = 10 mm.

2438 The Plant Cell

genetic inactivation of the indole glucosinolate biosynthesis/

metabolism pathway when the fungus switches more rapidly

from surface to invasive growth using a shortcut that bypasses

time-consuming appressorium development. Alternatively, the

apparent insensitivity ofCg entry attempts underneathmelanized

appressoria to presumed PEN2-derived antimicrobials could

result from the ability of the fungus to sequester and/or detoxify

such compounds on wild-type plants only in the presence of

mature appressoria. For example, theM. grisea ABC3 multidrug

resistance transporter exerts a critical role during early stages of

pathogenesis within appressoria, likely by imparting tolerance

against xenobiotic conditions encountered during pathogenic

development (Sun et al., 2006). Clearly, there must be extracel-

lular defense pathways other than PEN1-dependent exocytosis

and PEN2-dependent precursor activation of antimicrobials that

terminate nonadapted Cg entry attempts after appressorium

differentiation.

Many fungi infect both above-ground and below-ground plant

organs, of which the capacity to colonize roots is thought

to represent an ancestral trait (Dufresne and Osbourn, 2001;

Gunawardena and Hawes, 2002; Sesma and Osbourn, 2004;

Sukno et al., 2008). Although Colletotrichum is typically a foliar

pathogen,Colletotrichum graminicola has been shown to colonize

root tissue and to systemically infect above-ground stems and

leaves (Sukno et al., 2008). Likewise, M. oryzae, a well-studied

foliar pathogen, can also colonize roots and is capable of subse-

quent systemic invasion of above-ground tissues (Sesma and

Osbourn, 2004). The infection structures produced by C. gramini-

cola and M. oryzae during root colonization resemble those of

other root pathogenic fungi but are markedly different from those

seen during foliar colonization. For example, germinated spores

formed long “runner hyphae” on the surface of the root epidermis,

and entry into root epidermal cells was initiated by lateral swellings,

so-called hyphopodia, which emerge from these mature hyphae

(Sukno et al., 2008). Thus, while these previous studies and our

present work demonstrate the principal capacity ofColletotrichum

to enter plant cells of leaves and roots independently from ap-

pressorium differentiation, tip-based entry of conidial germ tubes

without appressoria on Arabidopsis leaves contrasts with the

hyphopodia-based entry into root cells. This points to the exis-

tence of a potential third pathway for host cell entry.

Genetic evidence for appressorium-independent entry into

Arabidopsis leaf cells was provided by the ability of nonadapted

C. orbiculare maf1 and mtk1 strains to form lesions on the pen2

plants with development of intracellular hyphae (Figure 8; see

Supplemental Figure 9 online). These mutant strains lack a MAPK

required for the early differentiation phase of appressorium for-

mation and a deduced MAPKKK presumably acting in the Maf1

MAPK pathway, respectively. Together, these findings suggest

that HTE is constitutively inhibited by the Maf1 pathway in wild-

type C. orbiculare. A constitutive suppression of fungal entry

attempts prior to appressorium formation is also supported by the

fact that essentially all germinated Colletotrichum spores differ-

entiate melanized appressoria on artificial surfaces such as glass

(Kojima et al., 2002). This excludes a requirement of host-derived

chemical cues for appressorium development and suggests

instead that Colletotrichum senses the nutritional status (such

as Glc or Suc) on leaf or root surfaces, which, together with other

Figure 9. The Two Nonadapted Colletotrichum Species Commonly

Take the Alternative Entry Mode at Wound Margins.

(A) Cg represses the formation of melanized appressoria at wound

margins of Arabidopsis leaves. Conidial suspensions of Cg expressing

GFP were inoculated on wounded leaves of the wild type and pen2

mutant without the addition of Glc. Inoculated plants were incubated for

14 h. When inoculated on wounded leaves, Cg repressed the formation

of melanized appressoria and formed colorless appressoria or elongated

hyphal structures, which is similar to the inoculation with Glc. The arrow

indicates a wounded site. Bars = 20 mm.

(B) The wild-type strain of C. orbiculare exhibits the maf1 phenotype at

wound margins of Arabidopsis. Conidial suspensions of the C. orbiculare

wild-type strain expressing GFP were inoculated on wounded leaves of

Arabidopsis wild-type plants. Inoculated plants were incubated for 14 h.

The wild-type strain of C. orbiculare repressed the formation of mela-

nized appressoria at the wound margins. Bars = 20 mm.

(C)HTE of two nonadaptedColletotrichum species at the woundmargins

of pen2 plants. Cg expressing RFP was inoculated on wounded leaves of

pen2 plants expressing PEN1-GFP. At 14 hpi, Cg developed intracellular

hyphae (ih) independently of a melanized appressorium at the wound

margins. The wild-type strain of C. orbiculare (Co) expressing GFP was

inoculated on the wounded leaves of pen2 plants. At 14 hpi,C. orbiculare

developed intracellular hyphae. Bars = 10 mm.

Fungal Entry Mode and Nonhost Resistance 2439

organ-specific cues, might override the default developmental

program leading to appressorium formation. Accordingly, sensing

of different microenvironments by Colletotrichum might trigger

hyphopodia differentiation in the rhizosphere or HTE on the leaf

surface by interference with the Maf1 developmental pathway.

Here, we have shown that the two tested nonadapted an-

thracnose fungi (Cg and C. orbiculare) predominantly initiate the

HTE morphogenetic response during pathogenesis at wound

margins on Arabidopsis leaves. It has been reported that some

Colletotrichum species, including Cg, develop lesions via myce-

lial inoculation on wounded surfaces of their hosts (Alkan et al.,

2008; Miyara et al., 2010). It is plausible that fungal hyphae enter

the hosts through thesewounds. However, our data suggest that

hyphal tips produced by the Colletotrichum sporelings also have

the ability to breach the cuticle of host cells directly at wound

margins. Although the identifiedmorphogenetic response switch

appears phylogenetically conserved, future experimentation is

needed to find out whether this switch (i.e., from turgor-mediated

invasion to HTE) has a selective advantage for the reproductive

success of the pathogen in nature. Irrespective of this, our

findings point to the existence of an unexpected fungal sensing

system to initiate markedly different morphogenetic responses

for pathogen entry on intact tissue and at wound sites. The

avocado isolates of Cg exhibit quiescent infection of unripe

avocado fruits but develop lesions during fruit ripening (Prusky,

1996). This phenomenon correlates with the breakdown of

antimicrobials during ripening. The finding that HTE becomes

effective on Arabidopsis lacking the PEN2 antimicrobial path-

way, which is enhanced by a Glc/Suc-rich microenvironment,

suggests a possible link between HTE and the Colletotrichum

infection strategy on fruits during ripening.

Plant cell penetration on leaf surfaces without appressorium

differentiation has recently been noted also for M. oryzae, al-

though this invasion mode was not further studied at the micro-

scopic level (Park et al., 2009). Thus, quantitative data resulting

from this alternative entry mode versus appressorium-depen-

dent penetration are unknown. Nonetheless, each of three M.

oryzae mutant strains, lacking genes required for appressorium

formation (Dcpka, Dpmk1, and MG01, in which the molecular

lesion is unknown), was able to infect Arabidopsis leaves, but

with reduced disease severity in comparison with wild-type M.

oryzae. Of note, these three M. oryzae mutants fail to infect and

colonize leaves of the natural host, rice (Oryza sativa; Park et al.,

2009), suggesting that the fungus can employ the alternative

entry route only on the nonhost Arabidopsis. Similarly, the C.

orbiculare maf1 and mtk1 strains are essentially nonpathogenic

upon conidiospore spray inoculation on leaves of the host plant,

cucumber (Kojima et al., 2002; see Supplemental Figure 8

online). Taken together, these findings suggest that appresso-

rium-independent invasion of leaf cells by these ascomycete

parasites has a more general significance, at least on nonhosts.

We do not know why the appressorium-deficient C. orbiculare

strains, defective in the Maf1 pathway, fail to colonize host

plants. However, it seems reasonable to assume that host-

adapted pathogens effectively suppress or avoid the activation

of preinvasion defense responses on their cognate hosts, while

on nonhosts such as Arabidopsis, preinvasion defense severely

restricts fungal entry (Collins et al., 2003; Lipka et al., 2005;

Shimada et al., 2006; Stein et al., 2006; this work). Thus, the

appressorium-deficient C. orbiculare strain might fail to block

effectively preinvasion defense responses on its host because

the pathogenesis route via appressoria is needed for effective

defense suppression.

In the necrotrophic pathogen Alternaria alternata, conidia have

a melanin layer, and this melanin is known to be required for UV

light tolerance of conidia (Kawamura et al., 1999). Although the

melanin layer contributes to the generation of turgor pressure in

appressoria of Magnaporthe and Colletotrichum species, it is

plausible that melanin has additional functions in the tolerance of

appressoria against environmental stresses such as UV light and

plant defense responses. Besides Colletotrichum and Magna-

porthe species, many fungal pathogens invade host plants by

using colorless appressoria (not melanized), suggesting that

melanization-independent entry modes represent ancestral

forms. Thus, in addition to the appressorium-independent tip-

based entry described in this report, Colletotrichum species

might have reutilized during evolution the deposition of melanin

in the program for appressorium formation to generate high

turgor and to be more tolerant against stresses.

Finally, the finding that pen2mutants retain nonhost resistance

against attack by melanized appressoria of Colletotrichum pre-

dicts the presence of unidentified factor(s) critical for defense

against this invasion style. Further studies on nonhost plant

defense responses againstColletotrichum species are expected

to reveal more molecular components that function in this

durable form of plant immunity.

METHODS

Fungal Strains and Media

Colletotrichum orbiculare (syn. Colletotrichum lagenarium) wild-type

strain 104-T (MAFF240422) was a stock culture of the Laboratory of

Plant Pathology, Kyoto University. The maf1 mutant DMA5 and the mtk1

mutant DMT1 were generated in the same laboratory (Kojima et al., 2002;

this study). The scd1 mutant SCD1REP1-1 was provided by Yasuyuki

Kubo (Kyoto Prefectural University; Tsuji et al., 2003). The Colletotrichum

gloeosporioides S9275 was provided by Shigenobu Yoshida (National

Institute for Agro-Environmental Sciences, Japan). Colletotrichum hig-

ginsianum isolate MAFF305635 was obtained from MAFF Genbank,

Japan. Cultures of the isolates were maintained on 3.9% (w/v) potato

dextrose agar medium (Difco) at 248C in the dark. MTK1was mutated

using an adaptation of a previously described in vitro transposon-tagging

procedure (Hamer et al., 2001). To mutate MTK1, a cosmid clone,

pCOMTK1, containingMTK1was used as the target. The gene-disruption

vector, pKOMTK1, was constructed by mobilizing a modified Tn7 trans-

posable element containing the hygromycin–phosphotransferase gene

cassette into pCOMTK1 in vitro. The transposon was inserted intoMTK1

at nucleotide 1150 (amino acid residue 345) in pKOMTK1. pKOMTK1was

transformed intoC. orbiculare strain 104-T to generate themtk1 knockout

mutants.

Plant Lines, Growth, and Inoculation

Seeds of Arabidopsis thaliana lines Col-0, pen1-1 (Collins et al., 2003),

pen2-1 (Lipka et al., 2005), pen2-2 (Lipka et al., 2005), cyp79B2 cyp79B3

(Zhao et al., 2002), cyp81F2-1 (Bednarek et al., 2009), pad3-1 (Zhou et al.,

1999), pen3-1 (Stein et al., 2006), pmr4-1 (Nishimura et al., 2003),

2440 The Plant Cell

pen3-1 sid2-1 (Stein et al., 2006), pen2-1with PEN1-GFP (this study), and

pen2-1 with PEN2-GFP (Lipka et al., 2005) were sown on rockwool,

treated at 48C in the dark for 2 d, and grown at 258C with 16 h of

illumination per day in nutrient medium. The pen2 plant expressing PEN1-

GFP was generated by crossing the pen2-1 plant with the pen1-1 plant

expressing PEN1-GFP (Collins et al., 2003; Lipka et al., 2005). For

macroscopic observation and trypan blue analysis,;4-week-old plants

were inoculated by spotting four 5-mL drops of conidial suspension (5 3

105 conidia per mL) on each leaf in the absence or presence of 0.1% Glc,

Suc, or sorbitol. For microscopic observation for invasion, ;12-d-old

plants were inoculated by spotting 2-mL drops of conidial suspension

(;5 3 105 conidia per mL) on each cotyledon. Inoculated plants then

were placed in a growth chamber at 258C with a cycle of 16 h of light and

8 h of dark and maintained at 100% RH. For analysis of carpropamid

treatment, conidia were inoculated in the presence of 10 mg/mL carpro-

pamid. In the inoculation assay on wounded Arabidopsis, leaves from

;12-d-old plants were wounded by a toothpick (toothpick treatment

indents the underlying plant tissue without perforation). Two-microliter

drops of conidial suspension were inoculated on the wounded area of

Arabidopsis leaves. The inoculation assay on cucumber (Cucumis sativus

cv Suyo) was performed as described previously (Takano et al., 2006). For

heat shock treatment, detached cotyledons of cucumber were dipped

into distilled water at 508C for 30 s. Conidial suspension of Cg was then

inoculated on the heat-treated cotyledons. As a control, cotyledons were

dipped into distilled water at 258C for 30 s before inoculation. The

inoculation assay onmulberry (Morus nigra) was performed by spotting of

Cg conidial suspension on leaves detached from mulberry plants (cv

Minamisakari) grown in the field.

Histochemical Staining

Plant cell death inducedby pathogen inoculation aswell as fungal primary

hyphae was investigated by staining with lactophenol–trypan blue and

destaining in saturated chloral hydrate (Koch and Slusarenko, 1990). To

determine the presence of callose deposits, samples were stained with

aniline blue as described by Adam and Somerville (1996). Material was

mounted on a slide in 50% glycerol and examined with a fluorescence

microscope (Zeiss Axioskop; Carl Zeiss) with Zeiss filter set 02 (excitation,

365 nm; dichroic, 395 nm; emission, 420 nm). The images were recorded

with a charge-coupled device camera (CoolSNAP; Roper Scientific).

Light Microscopy

To examine fungal behavior on Arabidopsis, plants were mounted in

water under a cover slip with the inoculated surface facing the objective

lens. In the case of inoculation with Glc, a portion of the conidia exhibits

germination at two sites at 14 hpi, which resulted in the formation of two

different structures, although the majority of conidia germinated at a

single site. In that case, the smaller class number among two structures

was counted by priority: a conidium forming a melanized appressorium

(class 1) and an elongated hypha (class 3) is categorized to conidium

forming melanized appressorium (class 1). The formation of invasion

hyphae into cucumber cotyledons was investigated as described previ-

ously (Asakura et al., 2009).

Confocal Microscopy

Detection of GFP and monomeric (m)RFP1 fluorescence was performed

using anOlympus Fluoview FV500 confocalmicroscopewith aNikon 403

or 603 PlanApo (1.4 numerical aperture) oil-immersion objective. Sam-

ples weremounted in water under a cover slip with the inoculated surface

facing the objective lens. The samples were excited with the argon laser

for GFP and with the HeNe laser for mRFP1. We used diachronic mirror

DM488/543, an SDM560 beam splitter, and two emission filters, BA505-

525 for GFP fluorescence detection and BA560IF for mRFP1 fluores-

cence detection.

Fungal Transformation

Preparation of protoplasts and transformation of C. orbiculare, Cg, and

C. higginsianum were performed according to a method described

previously (Takano et al., 2001). To visualize pathogens by expression

of themRFP1, pBATTEFPMRwas introduced into the tested pathogens

(Campbell et al., 2002; Asakura et al. 2009). Bialaphos-resistant trans-

formants were selected on regeneration medium containing bialaphos

(250 mg/mL). To visualize pathogens by expression of enhanced (E)

GFP, pTEFEGFP was introduced into the tested pathogens with

pCB1636 carrying a hygromycin-resistant gene (Sweigard et al.,

1997; Vanden Wymelenberg et al., 1997). Hygromycin-resistant trans-

formants were selected on regeneration medium containing hygro-

mycin (100 mg/mL).

Quantitative RT-PCR

Total RNAwas isolated from at least six leaves from six different plants for

each treatment by using the RNeasy Plant Mini kit (Qiagen) and treated

with DNase (Promega RQ1 RNase-free) to remove DNA contamination.

cDNA was synthesized with the PrimeScript RT reagent kit (Takara Bio).

Quantitative RT-PCR was performed with SYBR Premix Extaq (Takara)

and specific primers (see Supplemental Table 1 online). Quantitative

analysis of each mRNA was performed using the Thermal Cycler Dice

real-time system TP800 (Takara). ACTIN2 was used as a control for

normalizing the amount of cDNA.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative data library using the following locus identifiers:PEN1, At3g11820;

PEN2, At2g44490; PEN3, At1g59870; CYP79B2, At4g39950; CYP79B3,

At2g22330; CYP81F2, At5g57220; PAD3, At3g26830; PMR4, At4g03550;

ACTIN2, At3g18780. Other data can be found in the GenBank/EMBL data

libraries under the following accession numbers:MTK1, AB544078;MCK1,

XM_368361; SCD1, D86079.

Supplemental Data

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

Supplemental Figure 1. Trypan Blue Staining of Leaves Inoculated

with Adapted and Nonadapted Colletotrichum Species.

Supplemental Figure 2. The Effects of Sorbitol and Suc on Patho-

genicity of Cg on pen2 Mutant Plants.

Supplemental Figure 3. The Adapted C. higginsianum Develops

Melanized Appressoria in the Presence of Glc.

Supplemental Figure 4. Standard Cg Infection on pen2 Plants in the

Presence of Carpropamid.

Supplemental Figure 5. Plasmolysis Assay on Arabidopsis Cells

Invaded by Cg via HTE.

Supplemental Figure 6. Cg Develops Intracellular Hyphae under-

neath Melanized Appressoria on the Host Plant Mulberry.

Supplemental Figure 7. Amino Acid Sequence of C. orbiculare Mtk1.

Supplemental Figure 8. The mtk1 Mutant Exhibits Reduced Patho-

genicity.

Supplemental Figure 9. Inoculation Assay of C. orbiculare Pathoge-

nicity Mutants on the Arabidopsis pen2 Mutant.

Fungal Entry Mode and Nonhost Resistance 2441

ACKNOWLEDGMENTS

We thank Roger Y. Tsien for the mRFP1 gene and John Andrews

for pTEFEGFP. We also thank Shigenobu Yoshida (Cg isolate

S9275), Yasuyuki Kubo (the scd1 mutant), and the Ministry of Agricul-

ture, Forestry, and Fishers Genebank (C. higginsianum isolate

MAFF305635) for providing fungal strains. We are thankful to Masatoshi

Ichida for providing mulberry leaves. Thanks to Shauna Somerville

(pen3-1 and pen3-1 sid2-1) and the ABRC at Ohio State University

(pad3-1 and pmr4-1) for providing seeds. We thank Richard O’Connell

for critically reading the manuscript. This work was supported in part by

a Grant-in-Aid for Scientific Research (20380027) from the Ministry of

Education, Culture, Sports, Science, and Technology of Japan (Y.T.)

and by the Industrial Technology Research Grant Program in 2006 from

the New Energy and Industrial Technology Development Organization of

Japan (Y.T.).

Received January 31, 2010; revised June 1, 2010; accepted June 14,

2010; published July 6, 2010.

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Fungal Entry Mode and Nonhost Resistance 2443

DOI 10.1105/tpc.110.074344; originally published online July 6, 2010; 2010;22;2429-2443Plant Cell

Okuno, Paul Schulze-Lefert and Yoshitaka TakanoKei Hiruma, Mariko Onozawa-Komori, Fumika Takahashi, Makoto Asakura, Pawel Bednarek, Tetsuro

Resistance against Anthracnose PathogensDependent Function of an Indole Glucosinolate Pathway in Arabidopsis for Nonhost−Entry Mode

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Supplemental Data /content/suppl/2010/06/16/tpc.110.074344.DC1.html

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