Tang et al., Plant Physiology 2007 1 Running title: LACS2 ... · Tang et al., Plant Physiology 2007...

Tang et al., Plant Physiology 2007 1

Running title: LACS2, cutin, and disease resistance in Arabidopsis

Research Area: Plants Interacting with Other Organisms

*For correspondence: Dingzhong Tang, The State Key Laboratory of Plant Cell and

Chromosome Engineering, Institute Genetics and Developmental Biology, Chinese Academy of

Sciences, Beijing 100101, China.

Phone: 86-10-6484-7489; fax: 86-10-6484-7489; email: [email protected]

Plant Physiology Preview. Published on April 13, 2007, as DOI:10.1104/pp.106.094318

Copyright 2007 by the American Society of Plant Biologists

www.plantphysiol.orgon August 13, 2020 - Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.

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Mutations in LACS2, a long chain acyl-CoA synthetase, Enhance

Susceptibility to Avirulent Pseudomonas syringae, but Confer

Resistance to Botrytis cinerea in Arabidopsis1

Dingzhong Tang 2*, Michael T. Simonich3 and Roger W. Innes

Department of Biology, Indiana University, Bloomington, IN 47405, USA




1This work was supported by the National Institutes of Health (grant numbers R01 GM63761

and R01 GM046451 to R.W.I.). M.T.S. was supported by an NIH training grant in genetics (GM

07757).

2Present address: The State Key Laboratory of Plant Cell and Chromosome Engineering,

Institute Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,

China.

3Present address: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-6512

*Corresponding author; email [email protected]; fax 86-10-6484-7489.

The author responsible for distribution of materials integral to the findings presented in

this article in accordance with the policy described in the Instructions for Authors

(www.plantphysiol.org) is: Dingzhong Tang ([email protected])




Abstract

We identified an Arabidopsis thaliana mutant, sma4 (symptoms to multiple avr genotypes 4),

that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas

syringae pv. tomato, although bacterial growth is only moderately enhanced compared to wild-

type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal

pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a

necrotrophic fungal pathogen Botrytis cinerea. Germination of B. cinerea spores on sma4

mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4

mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower

humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes

LACS2, a member of the long chain acyl-CoA synthetases. LACS2 has previously been shown

to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants

for resistance to B. cinerea, att1 (for aberrant induction of type three genes),bodyguard and

lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant

cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the

pathogenesis of B. cinerea.




Disease resistance in plants is mediated by both preformed and induced defenses.

Examples of preformed defenses include physical barriers such as the cutin layer on leaf and

stem surfaces, antimicrobial enzymes stored in the tonoplast that are released upon cell damage,

and secondary metabolites that are often present at low constitutive levels, but whose levels are

increased during pathogen attack (Morrissey and Osbourn, 1999). Induced defenses include

production of reactive oxygen species, secretion of antimicrobial proteins, cross-linking of cell

walls, production of additional secondary metabolites, and the hypersensitive response (HR, a

type of programmed cell death) (Glazebrook, 2005). Rapid induction of defenses is usually

mediated by disease resistance (R) genes, which encode proteins that detect specific pathogen

virulence proteins (Jones and Dangl, 2006). The hallmark of R gene-mediated resistance is

specificity. Most R proteins detect only one or two virulence proteins. However, different R

genes activate very similar responses, suggesting that there are convergent points in R protein

signaling. Despite this seeming convergence, we have a very poor understanding of what

proteins function downstream of R proteins to activate the HR and other defense responses.

A large number of R genes from plants and their cognate virulence genes from pathogens

have been cloned. In Arabidopsis thaliana, the R genes RPM1 and RPS2 have been intensively

studied (Bent et al., 1994; Grant et al., 1995; Warren et al., 1998). RPM1 mediates detection of

at least two different pathogen virulence proteins, AvrB and AvrRpm1 (Bisgrove et al., 1994),

while RPS2 confers recognition of AvrRpt2 (Kunkel et al., 1993). In an attempt to identify genes

that function downstream of R gene activation, we designed a forward genetic screen to identify

Arabidopsis mutants that became susceptible to a Pseudomonas syringae pv. tomato strain that

carried both avrRpt2 and avrB (Pst DC3000(avrRpt2, avrB)). The wild-type Arabidopsis variety

Col-0 contains both RPS2 and RPM1. We hoped to avoid recovering rpm1 or rps2 mutants,

since in theory, mutations in either gene alone should still be resistant to such a strain.

We expected to identify several classes of mutants from this screen. The first class of

mutants was expected to contain mutations in downstream signaling components that are shared

by both RPM1 and RPS2, such as positive regulators of defense responses, including

transcription factors and regulators of the HR. A second class of mutants was expected to be in

genes required for assembly and/or stability of R protein complexes such as SGT1 and RAR1




(Tornero et al., 2002). A third class of mutants was expected to contain mutations in genes that

generally make the plant more susceptible to both virulent and avirulent pathogens, such as

mutations in preformed defense systems or components of the salicylic acid signaling pathway.

As described below, we uncovered mutations in the first and third class. In this paper, we

describe characterization and cloning of enhanced susceptibility mutant sma4 (for susceptible to

multiple avr genotypes), which appears to belong to the third class. The sma4 mutant displayed

severe disease symptoms after infection with Pst DC3000 carrying avrRpt2 and avrB, either

together, or individually. Interestingly, this mutant was highly resistant to the necrotrophic

fungal pathogen Botrytis cinerea. We cloned the SMA4 gene by map-based cloning, and found

that SMA4 encodes a member of the long chain acyl-CoA synthetase family, LACS2, which has

previously been shown to function in cutin synthesis (Schnurr et al., 2004).

RESULTS

Identification of Arabidopsis Mutants Susceptible to a P. syringae Strain Expressing Both

avrB and avrRpt2

Approximately 16,600 ethylmethanesulfonate, diepoxybutane, and fast neutron

mutagenized M2 plants were screened for disease symptoms following inoculation with DC3000

harboring both avrB and avrRpt2 on separate plasmids. Nine putative mutants that displayed

strong symptoms were selected. M3 progeny of these 9 mutants were dip inoculated with strain

DC3000 expressing avrB or avrRpt2 individually. Three of the mutants retained resistance to

DC3000(avrB), but appeared fully susceptible to DC3000(avrRpt2). Allelism tests revealed that

these mutations were in fact in RPS2. Although this was an unexpected result at the time, we

now know that AvrRpt2 affects RPM1 function by eliminating RIN4 (Axtell and Staskawicz,

2003; Mackey et al., 2003), a plant protein required for RPM1-mediated resistance (Mackey et

al., 2002). Thus, rps2 mutants become susceptible to DC3000(avrRpt2, avrB) because AvrRpt2

eliminates RIN4, blocking RPM1 function.

M3 progeny of the remaining six mutants displayed disease symptoms when inoculated

with either DC3000(avrB) or DC3000(avrRpt2). Allelism tests of these 6 mutants indicated that

three of the mutations were allelic to ndr1 (Century et al., 1997). NDR1 is a membrane




associated protein required by several CC-NBS-LRR class R genes, including RPM1, RPS2 and

RPS5 (Century et al., 1995).

The remaining three mutations were not allelic to ndr1 or each other. We designated

these mutants sma1, sma3 and sma4. Here, we describe characterization of the sma4 mutant and

the cloning of the SMA4 gene. The other two mutants remain to be fully characterized.

The sma4 Mutation Confers Partial Susceptibility to Several Avirulent P. syringae Strains

Varying degrees of necrotic collapse were consistently observed on sma4 plants relative

to wild-type Col-0 by 3 to 4 days after dip inoculation with strain DC3000 carrying avrRpt2,

avrB (Fig. 1a, 1b) or avrPphB (data not shown). These observations suggested that the DC3000

avirulent genotypes were at least partially virulent on sma4 mutant plants.

The symptoms of necrotic leaf collapse in response to each of the Pst DC3000 avr

genotypes were very atypical compared to those observed when virulent DC3000 was allowed to

infect wild-type Col-0 plants. The sma4 symptoms in response to infection by strain

DC3000(avrRpt2) were the most severe. Typically, some necrotic collapse was observed as early

as 48 hrs after dip inoculation at 1.5 x 108 cfu/ml. By late day 3 and early day 4 after infection,

most of the older, outer rosette leaves were observed to be entirely collapsed and dead. In

contrast, a virulent Pst DC3000 infection on wild-type Col-0 plants typically produces chlorosis,

large water soaked lesions and pin-point necrotic pits over the surface of Arabidopsis leaves

(Whalen et al., 1991). Rarely is whole leaf collapse and death observed in a virulent Pst DC3000

interaction at an inoculum concentration of 1.5 x 108 cfu/ml. Strains of DC3000 carrying avrB,

avrRpm1 and avrPphB induced a phenotype on sma4 plants similar to that induced by DC3000

(avrRpt2). However, these responses were consistently less severe (Fig. 1 and data not shown).

Examination of sma4 plants inoculated with the same concentration of the virulent

control strain DC3000(avrB::Ω) (Simonich and Innes, 1995) consistently showed symptoms of

chlorosis, water soaked lesions and necrotic pits, similar to wild-type Col-0. This indicated that

sma4 plants could develop classic symptoms of susceptibility. However, the disease symptoms

appeared more damaging to the sma4 plants than to Col-0 plants. Often, the sma4 mutant plants

died within 4 - 5 days of infection by strain DC3000(avrB::Ω), while identical inoculations of

wild-type Col-0 plants were never observed to kill the plants (data not shown). Interestingly,




although sma4 displayed more severe disease symptoms, there was no significant difference in

bacterial growth between Col-0 and sma4 plants 3 days after infection by strain

DC3000(avrB::Ω)(data not shown).

The sma4 phenotype of necrotic collapse more closely resembled a hypersensitive

response (HR) than true susceptibility. The HR is strongly associated with cessation of bacterial

growth and other resistant responses in the infected plant (Klement, 1982). To determine whether

the atypical symptoms observed on sma4 plants reflected susceptibility or an extreme

hypersensitive resistance, growth of two different DC3000 avirulent genotypes was monitored in

sma4 leaves. Figures 1c and 1d show that DC3000(avrRpt2) and DC3000(avrB) grew to higher

levels in sma4 leaves compared to wild-type Col-0 leaves, but this growth was not as high as that

of the virulent control strain DC3000(avrB::Ω). These results indicate that the sma4 mutant

phenotype is not the result of an enhanced HR. They also indicate that the sma4 mutation does

not block signaling by the RPM1 and RPS2 resistance genes, as growth of the avirulent strains is

still significantly reduced.

sma4 Leaves Are Intolerant of Weak Salt Solutions

We normally use a low (1 x 105 cfu/ml) concentration of pathogen carried in a solution of

10 mM MgCl2 with 0.001% L-77 wetting agent as the method of inoculation for monitoring in

planta pathogen growth. However, we noted that sma4 plants rapidly (within 2 hours) exhibited

severe necrotic leaf collapse after vacuum infiltrating pathogen cells suspended in a 10 mM

MgCl2 solution. Vacuum infiltration causes the leaves to become saturated with the infiltrated

solution in the intercellular leaf spaces. The plants are allowed to shed the excess water by

drying out slowly over a period of a few hours. This drying is occasionally observed to damage

some wild-type leaves along their margins where some necrosis may develop. However, sma4

plants consistently developed extensive tissue collapse on more than 50% of their leaves or were

entirely killed by the drying process. The collapse developed too quickly to be explained by a

pathogen-induced effect such as HR induction, which normally takes at least 12 hours to become

visible. We assessed whether the vacuum infiltration process, the subsequent leaf drying, or the

buffer components were the cause of this sma4 phenotype. Vacuum infiltration of sterile 10 mM

MgC12 and 0.001% L-77 caused the same degree of sma4 tissue collapse as was seen with the




pathogen inoculations. Vacuum infiltration with the pathogen carried in distilled water plus

0.001% L-77 did not cause any more sma4 leaf collapse than collapse of wild-type Col-0 leaves

within 2 hours of the infiltration. Finally, sma4 leaves vacuum infiltrated with distilled water

plus L-77 and no pathogen also did not display collapse. These observations suggested that the

10 mM MgCl2 was responsible for the leaf collapse. We therefore used distilled water instead of

10 mM MgCl2 to generate the bacterial growth curve data shown in Figure 1.

To ascertain whether this effect was limited to salt exposure or reflected a general

osmotic sensitivity conferred by the sma4 mutation, we repeated the vacuum infiltrations with

sterile 15 mM NaC1, 15 mM MgSO4, sterile 20 mM glucose and sterile 20 mM mannitol plus

0.001% L-77 wetting agent. The NaCl and MgSO4 treatments produced a response similar to the

MgCl2 treatments in severity, while the glucose and mannitol treatments responded similar to

infiltration with water. We also noted that we could block the observed collapse by loosely

sealing the salt infiltrated sma4 plants under plastic covers, which kept the surrounding humidity

relatively high. However, these covers did not allow the leaves to dry by shedding the excess

water from their intercellular spaces. These results suggested that the rapid tissue collapse

(within 2 hours) was triggered by salt exposure, but also required the drying process after salt

exposure. The phenotype was not attributable to a general osmotic sensitivity of the sma4 leaf

cells.

sma4 Seedlings Are Highly Sensitive to Lowered Humidity

We routinely germinate our Arabidopsis seeds in soil-filled pots under clear plastic

domes, which provides an environment of near 100% relative humidity. Approximately 1-2

weeks after germinating, when the first true leaves become visible, we remove the domes,

resulting in a rapid drop in humidity from about 100% to about 60%. Although wild-type

seedlings tolerate this sudden humidity drop without visible effects, sma4 seedlings were

observed to undergo a high rate of necrosis and death (Fig. 2), presumably due to desiccation of

the seedlings. Mutant sma4 seedlings up to 2.5 weeks post germination consistently suffered leaf

collapse within 2 hours after removal of the humidity cover. The sma4 leaf collapse could be

blocked by leaving the seedlings under the humidity cover for an additional 1.5 - 2 weeks. At




this age, removal of the humidity cover still resulted in some rapidly appearing necrosis on sma4

leaf margins, but leaf death rarely occurred.

sma4 Leaf Cells Leak Ions Faster than Wild-type Col-0 Cells During the HR and After Salt

Exposure

To further assess the relationship of the sma4 mutation to pathogen susceptibility, salt

stress and desiccation sensitivity, we compared the rate and amount of ion leakage from sma4

and Col-0 cells as a possible indicator of cell integrity during the HR and after vacuum

infiltration of 10 mM MgC12 and MgSO4. We inoculated individual leaves of sma4 mutant and

wild-type Col-0 plants with PsgR4(avrRpt2) at a cell density of 5 x 107 cfu/ml suspended in

distilled water. We assayed the amount of ion leakage every five hours for 25 hours by

measuring the conductivity of fluid eluted from sampled leaves. Figure 3a indicates that by 12

hours after inoculation, sma4 leaf cells had leaked a significantly greater amount of ions than the

Col-0 leaf cells had. By 24 hours, the point at which a visible HR is plainly visible on both sma4

and Col-0 leaves, the difference was even more dramatic. We repeated this experiment with

strain Pst DC3000(avrRpt2) infiltrated at a concentration of 1 x 107 cfu/ml and obtained a similar

result (not shown). These results suggested that, while sma4 plants develop an HR that is visibly

and microscopically similar to Col-0 plants, the sma4 leaves appear to leak cell contents faster

than Col-0 leaves in response to an HR-inducing bacterial strain.

We also assessed the effect of vacuum infiltration of 10 mM MgC12 and 10 mM MgSO4

on ion leakage (Fig. 3b). The rapid collapse of sma4 leaves correlated with a greater degree of

ion leakage from sma4 leaves after salt treatment compared to Col-0 leaves (Fig. 3b).

sma4 Plants Display Enhanced Disease Resistance to Botrytis cinerea

Because sma4 mutant plants appeared to be more prone to leaf collapse after both biotic

and abiotic stresses, we hypothesized that the mutant would display enhanced sensitivity to a

necrotrophic pathogen such as Botrytis cinerea, which actively kills infected leaves and grows on

the dead tissue. Wild type Col-0 plants are moderately susceptible to this pathogen, and growth

of B. cinerea can be enhanced by induction of an HR (Thomma et al., 1998; Govrin and Levine,




2000). Contrary to our expectations, sma4 mutant leaves displayed enhanced resistance to B.

cinerea (Fig. 4). To quantify resistance we inoculated detached leaves with a drop of spore

suspension, then measured lesion size five days after inoculation. By this time point, lesions in

wild-type Col-0 leaves had spread across the entire leaf. In sma4 plants, however, the lesions

were still confined to a small area (Fig. 4a, 4b). To gain additional insight into this enhanced

resistance to B. cinerea, we performed trypan blue staining to examine fungal growth. Figure 4c

shows that by day 3 in wild type Col-0 leaves, the fungus formed extensive hyphae that ramified

throughout the mesophyll. In contrast, on sma4 leaves, many of the spores failed to germinate,

and those that did failed to penetrate the epidermal cells and ceased to elongate (Fig. 4d).

In addition to B. cinerea, we also tested a biotrophic fungal pathogen, Erysiphe

cichoracearum strain UCSC, which is virulent on wild-type Col-0 plants (Adam and Somerville,

1996) and another necrotrophic fungal pathogen Alternaria brassicicola. We observed no

significant difference between Col-0 wild type and the sma4 mutants in their response to these

pathogens (data not shown).

sma4-Mediated Resistance to B. cinerea Is COI1 and EIN2 Independent

Resistance to necrotrophic pathogens is often dependent on JA and ethylene signaling

pathways (Glazebrook, 2005). To assess whether the JA/ethylene pathways contribute to sma4-

mediated B. cinerea resistance, we conducted double mutant analysis using mutations in COI1

and EIN2, central components of the JA and ethylene pathways. The sma4-mediated resistance

was only slightly suppressed by the coi1 and ein2 mutations (Fig. 5a,b), indicating that sma4-

mediated resistance to B. cinerea does not require intact JA and ethylene pathways. We also

tested coi1/sma4 and ein2/sma4 for responses to Pst DC3000(avrRpt2). As shown in Figure 5c,

the coi1 mutation suppressed the sma4-mediated susceptible phenotype. This observation was

consistent with the notion that JA and SA pathways are mutually antagonistic (Kunkel and

Brooks, 2002). According to this hypothesis, the SA signaling would be enhanced while the JA

pathway is suppressed in a coi1/sma4 mutant. The increased SA signaling would limit bacterial

growth and lead to suppression of the sma4-mediated susceptibility phenotype in response to Pst

DC3000(avrRpt2). In contrast, the ein2 mutation had no effect on sma4-mediated susceptible




phenotypes to Pst DC3000(avrRpt2), indicating that the ethylene pathway does not play a

significant role in sma4-mediated disease phenotypes.

Genetic Mapping and Identification of SMA4

To characterize the genetic basis of sma4 mediated-responses to pathogens, the mutant

was back-crossed to wild-type Col-0. The Fl progeny were resistant to dip inoculation with

DC3000 (avrRpm1), which indicated that sma4 was a recessive mutation. Consistent with this,

the F2 progeny segregated 3:1 wild-type to sma4 mutant phenotypes. To verify that partial

susceptibility to multiple avr genotypes was indeed conferred by a single mutation, several F3

families from the sma4 x Col-0 F2 progeny were tested for symptoms after dip inoculation with

DC3000(avrRpml), DC3000(avrB) and DC3000(avrRpt2). These F3 families displayed

symptoms of partial susceptibility to each of the different avr genotypes, indicating that the sma4

phenotype was conferred by a single mutant locus.

Using PCR-based molecular markers, we mapped the sma4 mutation to an 88 kb interval

on chromosome I (see Materials and Methods). We then amplified and sequenced open reading

frames from the sma4 mutant from most of the predicted genes in this region, except for

pseudogenes and genes encoding tRNA, or ribosomal RNA. In total, 15 genes were sequenced

and a T to A mutation was found in At1g49430. No mutation was found in the other genes

sequenced in this region. The point mutation in the sma4 mutant was located in the left border of

intron 4 of At1g49430 according to the annotated sequence of this gene (Fig. 6a). To examine

whether this mutation affected the splicing of At1g49430, we performed reverse transcriptase-

mediated polymerase chain reaction (RT-PCR) followed by direct sequencing the cDNA of this

gene from both sma4 and Col-0 plants. These analyses revealed that intron 4 was retained in the

sma4 cDNA, resulting in a 145 base insertion. This insertion caused a premature stop codon,

which would prevent translation of the last 15 exons of SMA4 (Fig. 6a), thus the sma4 mutation

likely causes a complete loss of function.

To further confirm that SMA4 is At1g49430, we complemented the sma4 mutant with a

6.8kb genomic DNA sequence, containing the full-length At1g49430 gene including 1.5 kb of

upstream promoter region and 0.8 kb of down stream sequences. Figure 6b and 6c show that this




genomic DNA fragment rescued sma4 mediated-disease phenotypes. These data demonstrated

that At1g49430 is the SMA4 gene.

SMA4 Encodes an Acyl-CoA Synthetase

At1g49430 has previously been shown to encode a long-chain acyl-CoA synthetase

(LACS), of which there are 9 family members in Arabidopsis (Shockey et al., 2002).

Specifically, At1g49430 encodes LACS2, which has been shown to be involved in cutin

biosynthesis (Schnurr et al., 2004). We tested lacs2-1, a T-DNA insertion allele (Schnurr et al.,

2004), for its response to Pst DC3000(avrRpt2) and found that it displayed a sma4-like

susceptible phenotype (Fig. 6b). We also tested the lacs2-1 mutant for resistance to B. cinerea

and found it to display sma4-like enhanced resistance (Fig. 5b), confirming that these phenotypes

in the sma4 mutant are likely caused by the loss of LACS2 function.

It should be noted that the lacs2-1 mutant has been reported to display a strong dwarfed

phenotype with small wrinkled leaves (Schnurr et al., 2004), which we did not observe in the

sma4 mutant. Under our growth conditions, however, lacs2-1 plants were only slightly smaller

than sma4 and wild-type plants (Fig. 6B and data not shown), suggesting that the dwarfed

phenotype reported by Schnurr et al. (2004) is influenced by environmental conditions such as

day length, light intensity and relative humidity. The slight decrease in plant size that we did

observe in the lacs2-1 mutant might be caused by differences in genetic background, as the

lacs2-1 mutant was isolated in the Col-0 glabrous-1 mutant background while sma4 was isolated

in the Col-0 wild-type background.

Several different Arabidopsis mutants with altered cuticle structure display enhanced

resistance to B. cinerea

To gain more insight into the role of plant cuticle structure in resistance to B. cinera, we

tested three additional cutin-defective mutants for resistance to B. cinerea, att1 (for aberrant

induction of type three genes), bodyguard (bdg) and lacerata (lcr). (Xiao et al., 2004;

Kurdyukov et al., 2006; Wellesen et al., 2001). Consistent with the sma4 results, all three

mutants showed increased resistance to B. cinerea (Fig. 7), indicating that a loss of cuticle




integrity in general leads to enhanced resistance, rather than a specific structural change in the

sma4 mutant.

DISCUSSION

We have shown that loss of LACS2 function makes Arabidopsis more susceptible to

avirulent strains of the Arabidopsis bacterial pathogen, Pst DC3000, but more resistant to a

virulent strain of the necrotrophic fungus B. cinerea. Why does loss of LACS2 function cause

these phenotypes? LACS2 encodes a long-chain acyl-CoA synthetase (Schnurr et al., 2004).

LACS enzymes activate free fatty acids to acyl-CoAs, a key step for the utilization of fatty acids

by most lipid metabolic enzymes (Shockey et al., 2002). LACS2 is expressed in the epidermal

cell layer of Arabidopsis leaves and the T-DNA insertion mutant lacs2-1 has a thinner cuticle

layer compared to wild-type plants. A preferred substrate of the LACS2 enzyme is ω-

hydroxypalmitic acid, thus Schnurr and colleagues concluded that LACS2 catalyzes the synthesis

of ω-hydroxy fatty acid-CoA intermediates required for cutin synthesis (Schnurr et al., 2004).

Leaves of a lacs2-1 null mutant release chlorophyll faster than wild-type leaves when immersed

in 80% ethanol and support pollen germination, which indicates that the cuticular barrier on

lacs2-1 plants is more permeable (Schnurr et al., 2004). We propose that it is this increased

permeability that causes the enhanced tissue collapse upon infiltration of dilute salt solutions or

avirulent pathogens, and causes enhanced resistance to B. cinerea.

Support for this hypothesis comes from work on ATT1, which encodes an Arabidopsis

cytochrome P450 monooxygenase (CYP86A2) that is required for proper cuticle development.

Loss-of-function att1 mutants have a lower cutin content, a loose cuticle ultrastructure, and

increased rates of water vapor transmission (Xiao et al., 2004). Because cutin also lines the

substomatal chamber in Arabidopsis leaves, this change in ultrastructure likely allows more rapid

leakage of cell fluids into this area, which is a primary point of colonization during P. syringae

infection. An increase in water flow into the substomatal chamber would be expected to increase

the growth of P. syringae strains as water stress is believed to be a limiting factor during P.

syringae infections (Wright and Beattie, 2004). Consistent with this hypothesis, and similar to

the sma4 mutant, the att1 mutant displays enhanced disease symptoms, including leaf collapse,

when inoculated with Pst strain DC3000. Furthermore, Xiao et al. showed that the expression of




the P. syringae virulence genes avrPto and hrpL is greatly enhanced in the substomatal chambers

of the att1 mutant compared to wild-type plants. This observation suggests that the att1 mutant

produces an eliciting signal that accumulates in the substomatal chamber more readily than in

wild-type plants, or alternatively, fail to produce a signal that suppresses induction of these

genes. The former hypothesis is consistent with a reduction in the cuticular barrier lining the

substomatal cavity.

An increase in cuticle permeability to water vapor in the sma4 mutant is consistent with

our observation that sma4 seedlings are quite sensitive to rapid decreases in humidity (Fig. 2).

An increase in cuticle permeability would also explain the observed increase in ion leakage after

bacterial infection or under salt exposure (Fig. 3).

The most interesting phenotype of sma4, however, is its resistance to the necrotrophic

fungus B. cinerea. Given that sma4 leaves collapse and die more rapidly when infected with P.

syringae, an increased resistance to a necrotroph was unexpected. One possible explanation for

the enhanced resistance is that B. cinerea may rely on specific physical and/or chemical queues

on the leaf surface to promote germination and penetration. Host surface structure has been

implicated in the pathogenesis of other necrotrophic pathogens. For example, in the interaction

between Collectotrichum trifolii and alfafa, the host surface chemistry appears to be important

for induction of fungal gene expression required for pathogenic development (Dickman et al.,

2003). Under this scenario, because of the inappropriate host cuticle structure in the sma4

mutant, B. cinerea is unable to induce gene expression required for pathogenic development. As

a result, the spores fail to penetrate the host surface and ultimately, fail to colonize and cause

disease in the host plants. Arguing against this hypothesis, however, is that B. cinerea has a very

broad host range, infecting over 200 plant species, which likely have different surface

chemistries. In addition, B. cinerea is routinely cultivated on potato dextrose agar, on which

spores readily germinate, indicating that B. cinerea does not have special requirements for

germination, whereas germination on sma4/lacs2 mutant leaves was poor. This latter

observation suggests that something on sma4/lacs2 mutant leaves may actively inhibit

germination and hyphal growth.

If the inhibition hypothesis is correct, it is plausible that the permeability of the cuticle

enhances export of an antifungal compound to the leaf surface, or alternatively, enhances import

of a fungal elicitor that triggers production of an antifungal compound. Evidence that export of




antifungal compounds to the leaf surface is important comes from recent work on the

Arabidopsis PEN3 gene. PEN3 encodes a plasma-membrane localized ATP-binding cassette

transporter that localizes around fungal penetration sites (Stein et al., 2006). Loss of PEN3

function causes increased susceptibility to the necrotrophic fungus Plectosphaerella cucumerina

(Stein et al., 2006), implying that export of an unidentified compound contributes to resistance to

this fungus. However, pen3 mutants do not display enhanced susceptibility to B. cinerea,

possibly because wild-type Arabidopsis is already quite susceptible. Further evidence that

increases in cuticle permeability leads to enhanced resistance to B. cinerea comes from our

finding that three different cuticle-defective mutants all display enhanced resistance to B. cinerea

(Fig. 7). These mutants have different structural changes in their cutin, but all have increased

permeability (Wellesen et al., 2001; Xiao et al., 2004; Kurdyukov et al., 2006).

It has recently been reported that a transgenic Arabidopsis line that expresses a fungal

cutinase displays enhanced resistance to B. cinerea (Chassot and Métraux, 2005; Chassot et al.,

2007). Similar to lacs2 mutants, the cutinase transgenic lines form a defective cuticle that has

increased permeability (Sieber et al., 2000). Most significantly, these transgenic lines, as well as

the bdg mutant, were shown to release a fungitoxic activity from the surface of their leaves that

was not released by wild-type leaves (Chassot et al., 2007). Thus, the enhanced resistance to B.

cinerea observed in sma4 plants is likely due to this same antifungal activity. This supposition

has very recently been confirmed by Bessire et al. (2007), who have shown that diffusates from

lacs2 mutant leaves contain a strong antifungal activity. These observations suggest that

Arabidopsis produces a compound that is very toxic to B. cinerea, but which is limited in its

effectiveness either by its ability to reach the leaf surface in a timely manner, or to be induced in

a timely manner. Increasing the permeability of the cuticle layer, by any means, enhances its

induction and/or release, thus conferring resistance.

MATERIALS AND METHODS

Plant Growth Conditions and Mutant Screening

Arabidopsis thaliana plants were grown in growth rooms under a 9 hr-light/15 hr-dark

cycle at 23˚C as described previously (Frye and Innes, 1998). Approximately 16,600




ethylmethanesulfonate, diepoxybutane, and fast neutron mutagenized Col-0 plants (M2

generation) were inoculated with P. syringae pv. tomato strain DC3000 carrying both avrB (on

plasmid pDSK600) and avrRpt2 (on plasmid pVSP61) and scored for disease responses 3 days

after inoculation. Plants displaying severe disease phenotypes were selected and allowed to set

seeds. The sma4 mutant was backcrossed twice to wild-type Col-0 prior to performing the

analyses presented in this paper, with the exception of the original bacterial growth data shown

in Figures 1c and 1d, which was performed on non-backcrossed material.

Infections with Pathogens

Inoculation of A. thaliana plants with P. syringae pv. tomato strain DC3000 and

measurement of bacterial growth within leaves was performed as described previously using

vacuum infiltration of 6 week old plants (Simonich and Innes, 1995), except that inocula were

prepared in distilled water rather than 10 mM MgCl2 to avoid the confounding effects of sma4

tissue damage caused by salt solutions. HR inoculations were performed by injecting bacterial

cells suspended in distilled water into the abaxial side of 6 week old leaves with a needleless 1

ml syringe. HR formation was assayed 24 hours after infiltration. Inoculation of A. thaliana

plants with B. cinerea was performed as described by Ferrari et al. 2003 using detached leaves

(strain obtained from F.M. Ausubel and described in (Ferrari et al., 2003). Fungal structures and

dead plant cells were stained by Trypan blue (Frye and Innes, 1998). Samples were observed and

photographed using a Nikon SMZ1500-dissecting microscope. E. cichoracearum strain UCSC1

was maintained and inoculated onto Arabidopsis plants as describe previously (Tang and Innes,

2002).

Ion Leakage Measurements

Intact leaves were entirely infiltrated with a bacterial density of 5 x 107 cfu/ml in

deionized water. At 0, 5, 10, 15, 20 and 25 hours after infiltration, a random sample of inoculated

leaves was excised from the plants and five leaf discs, each 7 mm in diameter, were cut from the

excised leaves with a cork borer and immediately floated, abaxial side down, in 6 mL of 0.001%

L-77 surfactant (Union Carbide) in water. The sampling was done in triplicate for each time-




point. Leaf discs were floated for 4 hours after which conductivity measurements of the bathing

solution were made with a Radiometer Copenhagen model CDM2f conductivity meter and

Radiometer Copenhagen CDC104 detector (The London Company, Cleveland, OH).

Genetic and Physical Mapping of sma4

F2 progeny of a sma4 cross to Landsberg erecta (Ler) were used to genetically map the

SMA4 gene. The F2 plants were inoculated with Pst DC3000(avrRpt2) and scored 3 days after

inoculation. Plants displaying a sma4 phenotype were used for mapping. Initially, the sma4

mutation was mapped to chromosome I between SSLP markers T27K12 and CIW1. To further

localize the SMA4 gene, we developed new markers at intervals between these two markers

using Monsanto Col-0 and Ler polymorphism data (marker data available upon request). 556

susceptible F2 plants representing 1112 meioses were scored, which enabled us to localize sma4

between two markers at positions 29kb and 117kb of the bacterial artificial chromosome (BAC)

clone F13F21 (Genbank accession AC007504), defining an 88 kb region that co-segregated with

the sma4 mutation.

Sequencing of Candidate Genes

Candidate genes were amplified by PCR from genomic DNA isolated from the sma4

mutant and from wild-type plants and directly sequenced. All sequencing reactions were

performed using BigDye Terminator Kits (Applied Biosystems, Foster City, CA) and separated

on an ABI 3730 automated DNA sequencer. In total, 15 genes were amplified from the sma4

mutant by PCR and directly sequenced. To obtain the SMA4 cDNA sequence, RNA was isolated

and first strand cDNA synthesis was performed as previously described (Tang et al., 2005).

Complementation of the sma4 Mutant

A full-length SMA4 genomic sequence, including the promoter region and 3’ untranslated

region was amplified from BAC F13F21 using the following primers: 5’-

AACCGCTAGCTTCCTTATAAAAAGTTAAAGAAAAAG-3’ and 5’-

AATTGGGCCCCGTATGAGAATGATTAGTTTAGTTGA-3’. The PCR product was digested




with ApaI and NheI and ligated with the binary vector pGreen0029 digested with ApaI and XbaI

(Hellens et al., 2000). This DNA sequence contains a full-length At1g49430 gene including 1.5

kb of sequence 5’ to the start codon and 0.8 kb of sequence 3’ from the stop codon.

The pGreen0029:At1g49430 construct was transformed into Agrobacterium tumefaciens

strain GV3101 by electroporation and selected on LB plates containing 50 µg/mL kanamycin

sulfate (Sigma). Arabidopsis plants were transformed using the floral dip method (Clough and

Bent, 1998). Transgenic plants were selected by growing on 0.5x Murashige and Skoog salts

plus 0.8% agar and 50 µg/mL kanamycin. Transformants were transplanted to soil 7 days after

germination and were inoculated with Pst DC3000(avrRpt2) and with B. cinerea when 5 weeks

old.

Construction of Double Mutants

Double mutants were created by standard genetic crosses. The ein2-1 mutation was in the

Col-0 background (Alonso et al., 1999), and coi1-1 was in the Col-6 background (Xie et al.,

1998). To identify the sma4 mutation in F2 progeny, we performed PCR amplification using

dCaps primers 5’-TATGTTATGACATGATCCAATCAATC-3’ and 5’-

AATTTTAGATATAGGTCAATTTTTTTGT-3’, followed by digestion with RsaI (New

England Biolabs, Beverly, MA).

ACKNOWLEDGEMENTS

We thank J. Browse for providing lacs2-1 seeds, J. Turner for providing coi1-1 seeds, J. Ecker

for providing ein2-1 seeds, J. Zhou for providing att1 seeds, A. Yephremov for providing bdg

and lcr seeds, F. M. Ausubel for providing the Botrytis cinerea strain, and T. Mengiste for

providing an Alternaria brassicicola strain. We also thank the Arabidopsis Biological Resource

Center at Ohio State for providing BAC clone F13F21.

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FIGURE LEGENDS

Figure 1. The sma4 mutant displays enhanced susceptibility to Pst DC3000 expressing avrRpt2 or

avrB. A and B, Six week old sma4 plants were dip inoculated with Pst DC3000(avrRpt2) (A) or Pst

DC3000(avrB) (B) and photographed 3 days later. C and D, Bacterial growth for Pst DC3000(avrRpt2)

(C) or Pst DC3000(avrB) (D) was monitored over a 4-day time course. Each data point represents the

mean and standard error of three samples. CFU, colony-forming units.

Figure 2. The sma4 mutant is highly sensitive to lowered humidity. Wild-type Col-0 and sma4 plants

were grown in soil for 10 days after germination under a clear plastic dome to maintain near 100%

relative humidity. A, Seedlings photographed immediately after removing the dome. B, Seedlings

photographed two hours after the dome was removed.

Figure 3. Ion leakage from sma4 mutant leaves is enhanced. A, Col-0 and sma4 ion leakage in response

to P. syringae pv. glycinea Race 4 (avrRpt2). PsgR4(avrRpt2) was infiltrated into single leaves at a

concentration of 5 x 10 7 cfu/ml. Five leaf discs were floated abaxial side down in deionized water for 4

hrs after which conductivity of the solution was measured in micro-ohms. Each data point is the mean

plus/minus the standard error, of three samples. B, sma4 leaves leak ions faster than wild-type Col-0

leaves after salt exposure. Ion leakage from sma4 and Col-0 leaves was measured after vacuum

infiltration with MgCl2 or MgSO4 solution.

Figure 4. The sma4 mutant displays enhanced disease resistance to B. cinerea. A, Leaves from six week

old Col-0 and sma4 plants were detached and placed in petri dishes, and inoculated with B. cinerea.

Leaves were photographed 5 days after inoculation. B, Lesion size induced by B. cinerea. Lesion size

was determined by measuring the major axis of the necrotic area at 5 days after inoculation. Bars

represent the mean and standard deviation from 8 samples. C and D, Leaves from Col-0 and sma4 plants

were stained with trypan blue and photographed 3 days after infection with B. cinerea.

Figure 5. Resistance to B. cinerea does not require COI1 or EIN2. A, Leaves from six-week old plants

were detached and placed in Petri dishes and inoculated with B. cinerea. Representative leaves were

photographed 3 days after inoculation. B, Lesion size induced by B. cinerea was measured 3 days after




inoculation. Bars represent the mean and standard deviation from eight samples. C, Six week old plants

were dip inoculated with Pst DC3000(avrRpt2). Plants were photographed 3 days after inoculation.

Figure 6. Structure of the SMA4 gene and complementation of the sma4 mutation. A, Exon and intron

structure of SMA4. The exon regions are indicated with rectangles and intron regions with lines. The

sma4 mutation is a point mutation in intron 4. The letters show nucleotide sequences from the beginning

of intron 4. The T to A mutation in sma4 is indicated by an uppercase letter. B and C, Complementation

of the sma4 mutation by transformation. Plants were dip inoculated with DC3000(avrRpt2) and

photographed 3 days after inoculation (B), or inoculated with B. cinerea and photographed 5 days after

inoculation (C).

Figure 7. The cutin defective mutants att1, bdg, and lcr display enhanced resistance to B.

cinerea. A, Leaves from five-week old plants were detached and placed in petri dishes, and

inoculated with B. cinerea. Leaves were photographed 3 days after inoculation. B, Lesion size

induced by B. cinerea. Lesion size was determined by measuring the major axis of the necrotic

area at 3 days after inoculation. Bars represent the mean and standard deviation from 8 samples.

















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