Identifi cation of a Small HeatShock Protein Associated 

with a Ras-Mediated 

Signaling Pathway in 

Ectomycorrhizal Symbiosis

Shiv Hiremath

Kirsten Lehtoma

Gopi K. Podila

United States

Department of 

Agriculture

Forest Service

Northern 

Research Station

Research Paper NRS-7
Abstract

Initiation, development, and establishment of a functional ectomycorrhiza involve 

a series of biochemical events mediated by a number of genes from the fungus 

as well as the host plant. We have identifi ed a heat-shock protein gene from 

Laccaria bicolor (Lbhsp) that appears to play a role in these events. The size 

and characteristics of Lbhsp suggest that it belongs to the family of small heatshock proteins described in the literature. Nucleotide sequencing of an almost 

full length cDNA indicated that the Lbhsp mRNA is about 611 nucleotides long 

and codes for a single protein of ~ 17 kDa. Isolation and characterization of the 

Lbhsp gene showed that it was made up of three exons separated by two small 

introns. Southern analysis suggested that the L. bicolor genome contains at least 

two copies of the Lbhsp gene. Temporal expression analyses revealed that the 

gene is expressed within 4 to12 hours after interaction with red pine roots. The 

yeast two-hybrid studies showed that the Lbhsp was closely associated with 

the ras gene (Lbras) described earlier. The data suggest that Lbhsp may play a 

supporting role in ras-mediated mycorrhizal signaling pathways during various 

stages of ectomycorrhizal development.

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Manuscript received for publication 10 October 2008
1

INTRODUCTION

Initiation, development, and establishment of a functional ectomycorrhiza 

involve a myriad of biochemical and genetic events in each partner that eventually 

culminate in the formation of a new functioning symbiotic organ (Duplessis et al. 

2005, Hilbert et al. 1991, Johansson et al. 2004, Martin and Tagu 1999, Martin 

et al. 2007, Nehls et al. 1999, Polidori et al. 2001, Voiblet et al. 2001). A number 

of dynamic signaling interactions take place between the host plant roots and the 

fungus leading to recognition of each other (Podila et al. 2002). If compatible, 

further changes in gene expression are initiated to bring about morphological 

changes needed for the development of the functioning ectomycorrhiza. Th us, 

early events in the interaction are crucial and determine the fi nal outcome. Th ey 

have been shown to involve activation of a cascade of molecular events in each 

partner. Because ectomycorrhiza represents a completely distinct new organ formed 

after the interaction, it is highly conceivable that genes related to cell growth and 

proliferation play an important role.

We identifi ed a gene Lbras from the ectomycorrhizal fungus Laccaria bicolor

that belongs to the ras family of genes found in other systems (Kim et al. 1998, 

Sundaram et al. 2001). Th e ras genes, present in a wide range of eukaryotes, 

including mammals and yeast, are involved in a variety of cellular signaling 

pathways controlling critical cellular events such as cell cycle progression and cell 

division (Broek et al. 1985, Ha et al. 2003, Rocha et al. 2001). Th e Lbras cDNA 

complemented ras2 function in Saccharomyces cerevisiae and had the ability to 

transform mammalian cells (Sundaram et al. 2001). Expression of Lbras, present 

as a single copy in the L. bicolor genome, was dependent upon interaction with 

host roots. Northern analysis showed that expression was detectable in the fungus 

as early as 12 to 24 hours after interaction with plant roots and continued to 

be expressed even in the established mycorrhizal tissue. Phylogenetic analysis 

with other ras proteins showed that Lbras is most closely related to Aras gene of 

Aspergillus nidulans.

Our examinations of early changes in gene expression in L. bicolor during its 

interaction with red pine roots indicated the presence of a heat-shock protein 

related gene whose expression followed a pattern similar to that of Lbras and other 

signal pathway related genes we have identifi ed. Th e similar pattern of expression as 

well as known relationships between heat-shock proteins and ras in other systems 

prompted us to undertake further studies to characterize the L. bicolor heat-shock 

protein (Lbhsp) gene. Th is protein was also identifi ed as an interacting protein with 

Lbras during yeast two-hybrid screening of a cDNA library from L. bicolor prepared 

after interaction with red pine seedling roots. We describe here the isolation and 

characterization of the Lbhsp cDNA, its gene, and determination of its expression 

pattern under diff erent physiological conditions. Th e data indicate that it is 

indeed a heat-shock protein gene and may have a role in ras-mediated signaling 

pathwaysfor eff ecting development and formation of the ectomycorrhiza.

Th e Authors

SHIV HIREMATH is a research 

biologist with the Northern 

Research Station in Delaware, OH.

KIRSTEN LEHTOMA is a 

biological laboratory technician 

with the Northern Research Station 

in Delaware, OH.

GOPI K. PODILA is a professor, 

Department of Biological Sciences, 

University of Alabama, Huntsville, 

AL.
2

MATERIALS AND METHODS

Media, Cultures, and Laccaria bicolorPinus resinosa Interactions

Th e Laccaria bicolor (Maire) Orton DR170 strain used 

for our studies was a basidiocarp isolate associated with 

the roots of red pine obtained from Michigan’s Upper 

Peninsula (provided by D. Richter, School of Forestry, 

Michigan Technological University, Houghton). Th e 

fungus was grown and maintained on MMN medium 

as previously described (Bills et al. 1995, Marx 1969). 

In vitro interactions and harvesting of interacted fungal 

tissue were carried out as described previously (Kim et al. 

1998, 1999). Synthesis of mycorrhizal tissue in soil was 

performed according to Bills et al. 1999.

Isolation of cDNA Clones Coding for 

Lbras Interacting Proteins

Isolation of total RNA from L. bicolor-P.resinosa 

ectomycorrhizal tissue was carried out as described 

previously (Kim et al. 1998). Th e Smart cDNA Synthesis 

Kit (Clontech, CA) was used to synthesize cDNA ligated 

with Sfi arms as per manufacturer’s instructions. LexA 

Match Maker Library kit (Clontech, CA) was used to 

perform the yeast two-hybrid screens as described (Fields 

and Song 1989, Sundaram et al. 2004).

For protein interaction studies, the mycorrhizal cDNA 

synthesized with the Smart cDNA kit was cloned 

into the Match Maker Library AD vector, full length 

Lbras cDNA was cloned into the Match Maker BD 

vector, and two-hybrid interactions were performed 

as per manufacturer’s instructions. A previously well 

characterized ras clone (Lbras) from the ectomycorrhizal 

fungus L. bicolor (Kim et al. 1998) was used as bait in 

a yeast two-hybrid interaction system. About 10,000 

colonies were screened for positive interactions by 

selection for Leu+ followed by a standard blue/white 

selection for expression of the β-galactosidase. Plasmids 

containing interacting clones were rescued from the yeast 

as per manufacturer’s instructions (Clontech, CA). E. 

coli DH5 alpha cells were transformed with the rescued 

plasmids, and resultant plasmid DNAs were isolated 

and subjected to further analysis. Selected clones were 

also tested for authenticity by one-on-one interaction 

to confi rm their interaction with Lbras. Th e strength 

of the interaction was determined using the liquid 

β-galactosidase assays (Pandey et al. 2006).

Sequence and Phylogenetic Analyses

Nucleotide sequencing was carried out on an ABI 

PRISM 310 Genetic Analyzer (Applied Biosystems, CA) 

and Dynamic ET sequencing kit (GE Health Systems, 

NJ). For sequence analyses, Sequencing Software version 

3.0 (PE Applied Bio-systems, CA) and MacDNAsis 

software version 5.0 (Hitachi Instruments, San Jose, 

CA) were used. Multiple alignments of the sequences 

were done with the Mutalin (www.toulouse.inra.fr). 

A parsimony-based phylogenetic analysis was done 

on hsp class proteins using the software PAUP version 

4.0b.8 (Sinauer Associates, MA) as previously described 

(Berlocher and Swoff ord 1997, Farris 1983, Sundaram et 

al. 2001).

Northern and Southern Analyses

Northern analyses were carried out essentially as 

described earlier (Kim et al. 1998). Total RNA from L. 

bicolor subjected to interaction with pine seedling roots 

for 12, 24, 36, 48, 72 and 96 hours was electrophoresed 

on agarose gels and transferred to Hybond-N 

membranes (GE Health Systems, NJ). Total RNA from 

free-living L. bicolor was used as control. Twenty μg 

RNA was loaded in each lane; gels were stained with 

SYBR Green, and the intensity of color was observed to 

confi rm equal amounts of RNA being present in each 

lane for comparisons.

Th e L. bicolor genomic DNA isolated from ground 

mycelia (Kim et al. 1998, Sundaram et al. 2004) was 

digested with restriction enzymes. About 10 μg genomic 

DNA was used for each restriction digestion. After the 

fragments were resolved by agarose gel electrophoresis, 

they were transferred to a Hybond-N+

 membrane. Th e 

cDNA fragment of Lbhsp labeled with 32P-dCTP using 

the Rediprime DNA Labeling Kit (GE Health Systems, 

NJ) served as a probe in Southern hybridization analysis 

(Kim et al. 1999).
3

RESULTS

Heat-shock Protein (Lbhsp) mRNA 

and Gene

An interaction-specifi c cDNA library, constructed using 

L. bicolor that had interacted with red pine roots for 4 to 

72 hours (Kim et al. 1998), was subjected to diff erential 

screening to identify cDNAs that show signifi cant 

changes in expression during early interaction (data not 

shown). One of the clones with an insert of ~ 600 bp 

was designated as the heat-shock protein cDNA (Lbhsp) 

after further characterization. Th e cDNA insert from 

this clone was subcloned and its complete nucleotide 

sequence was determined. It had 611 nucleotides without 

Figure 1.—Southern hybridization of L. bicolor genomic 

DNA for Lbhsp sequences. Hybridization of the genomic 

blot was done with Lbhsp-specifi c probe. Restriction 

enzymes used for digesting L. bicolor genomic DNA and 

position of molecular weight markers are indicated.

Figure 2.—The Lbhsp gene isolated from the L. bicolor genomic 

library. Complete nucleotide sequencing was done to identify the 

structure of the gene. Locations and sizes of introns and exons 

are indicated. The location of the initiation codon (ATG) and 

termination codon (Term) are also shown.

Exon

ATG 

193 bp 59 bp 221 bp 61 bp 198 

bp 

732 bp

Term

Intron

the poly(A) tail and encoded a single protein of 155 

amino acids (Genbank Database Accession Number 

AY117412). Th e predicted size of the protein was ~ 17 

kDa.

Southern analysis was performed using the Lbhsp cDNA 

insert as a probe on restriction enzyme digested L. bicolor

genomic DNA (Fig.1). Th e restriction enzymes used in 

the analysis did not have any sites in the cDNA used as 

the probe. As seen in the fi gure, the pattern suggested 

that at least two copies of the Lbhsp genes were present in 

the L. bicolor genome.

Th e cDNA insert was used as a probe to screen an L. 

bicolor genomic library to identify and isolate the Lbhsp

gene. Th e isolated gene was characterized by mapping 

with restriction enzymes, and its complete nucleotide 

sequence was determined (Fig. 2). Analysis of the 

nucleotide sequence showed that the Lbhsp gene is made 

up of three exons of 193, 221, and 198 bp separated by 

two small introns of 59 and 61 bp.

Comparison to known sequences deposited in the 

Genbank indicated that the nucleotide sequence and the 

derived amino acid sequence had signifi cant similarity 

to the small heat-shock proteins previously identifi ed 

in yeast and other organisms. Alignments showed 

signifi cant identity in conserved portions of the protein 

(Fig. 3). Phylogenetic analysis of closely related hsp16

sequences showed that the Lbhsp, while related to other 
4

hsp16 sequences, falls on a diff erent branch (Fig. 4). Th is 

suggests that Lbhsp may be functionally diff erent from 

other proteins in the hsp family.

Expression of Lbhsp

Expression analysis of Lbhsp gene was carried out for the 

free-living fungus (on agar plates) as well as for fungus 

interacting with the red pine roots (In vitro interaction 

system described earlier: Kim et al. 1999). For assays 

involving free-living fungal tissues, the fungus was fi rst 

grown on a cellophane membrane placed on MMN agar 

medium and later transferred to agar plates containing 

the interaction medium.

Northern analyses showed the presence of a ~ 600 b 

mRNA, which was consistent with the size predicted by 

the nucleotide sequence of the cDNA (Fig. 5). Th e Lbhsp

mRNA was not detectable in samples obtained from freeliving (agar plates) fungus (Lane Fr). However, samples 

from fungus that had interacted with the roots for 6 to 

72 hours showed the presence of this mRNA. Only traces 

could be detected in samples from the 6-hour interaction; 

however, after 12 hours it was clearly seen and its levels 

kept increasing until 72 hours. In other experiments we 

have detected mRNA as early as 4 hours after interaction 

by using more RNA in Northern analysis (data not 

shown). We also investigated the eff ect of 2.5 mM 

Figure 3.—Comparison of Lbhsp amino acid sequence to those in the Genbank. Comparison analysis indicated that 

the amino acid sequence had signifi cant similarity to heat-shock proteins previously identifi ed in the yeast and other 

organisms. Alignments showed signifi cant identity in conserved portions of the protein. The alignment with the amino 

acid sequence of the hsp 16 from Schizosaccharomyces pombe (Sp hsp16) is shown. The identities are shown with 

the amino acid marker and + indicates a similar amino acid. Other details such as total identities, similarities, and 

gaps are indicated.

Similarity to yeast heat-shock protein 

Score = 76.3 bits (186), Expect = 9e-14 

Identities = 44/110 (40%), Positives = 62/110 (56%), Gaps = 4/110 (3%) 

Sp hsp 16 LKPRMDLHEDKEKNLVTATFEFPGSKKEDVHLEIHNGRLVVSVE--NKISEEHDE 99 

 L P +D+HE K+ V+ E PG KKEDV + +G+L +S E N+ E E 

Lbhsp LSPSIDVHEGKDT--VSVDVELPGVKKEDVQVHYDSGKLTISGEVVNERKNESTE 87 

Sp hsp 16 SGYAVRERRYGKFSRTLQLPQGVKDDEIKAGMEDGVLTVTFPKSGAELAPKKISI 154 

 ERR+G FSRT+ +P + D I+A +G+LTVT PK K+I+I 

Lbhsp GNQRWSERRFGSFSRTITIPAKIDADRIEANFSNGLLTVTLPKVEKSQTKKQIAI 142 

glucose added to the interaction medium and found that 

it did not have any signifi cant eff ect on free-living and/or 

interacted tissues.

Protein-protein Interaction of Lbhsp

and Lbras

We used the yeast two-hybrid screening to determine the 

relationship between Lbhsp and Lbras. Screening the L. 

bicolor library using Lbras as bait, we were often able to 

isolate Lbhsp, suggesting that Lbhsp is closely linked and 

presumably has a role in the functioning of Lbras. Out 

of more than 50 positive yeast two-hybrid clones isolated 

as interacting partners of Lbras, Lbhsp was identifi ed 

more than six times. We repeated the experiment with 

individual clones (one-on-one) to confi rm that this 

association was indeed true. Liquid beta-galactosidase 

assays performed to determine the strength of the 

interaction showed that it is a very strong interaction.

DISCUSSION

Signifi cant changes in gene expression are initiated in L. 

bicolor during its interaction with red pine roots (Kim 

et al. 1998, Podila et al. 2002). We have identifi ed a 

number of genes that are turned on as early as 4 hours 

after interaction. Many of these were related to genes that 

have been shown to be involved in signal transduction 

events in other systems. Formation of a functioning 
5

ectomycorrhiza requires turning on of key genes that 

control events such as cell growth and proliferation. 

Th ese actions eventually lead to formation of a novel 

symbiotic organ. Th e Lbhsp gene reported here appears 

to belong to the same key group of genes involved in 

mediating cell growth and proliferation events.

To identify important genes involved in early interaction 

between L. bicolor and red pine roots, we used a 

diff erential screening procedure to generate a subset of 

cDNA clones that would represent an interaction-specifi c 

cDNA library. Th e genes in this library would be the 

ones whose expression is signifi cantly induced due to 

interaction with host roots. To achieve this, we made 

two cDNA probes using poly(A) RNA from L. bicolor

that had interacted with plant roots either for 0 hours or 

for 72 hours. We screened the cDNA library described 

above with both these probes separately. Comparison 

of the results from these screenings allowed us to select 

the cDNA clones that represented L. bicolor mRNA that 

was expressed only after interaction with the roots. Th is 

subset of clones was designated as the interaction-specifi c 

cDNA library, and some of these were subjected to 

further analyses.

Th e Lbhsp cDNA clone isolated represented an almost 

full length copy of the Lbhsp mRNA. Th e deduced 

protein from the nucleotide sequence showed signifi cant 

homology to the small heat-shock proteins described 

in the genome database. Particularly, Lbhsp protein was 

very similar to the yeast hsp16 protein. Southern analysis 

showed the presence of at least two copies of the Lbhsp

gene in the fungal genome. Th is is consistent with the 

recent fi nding of a single copy found in the haploid strain 

H82 of L. bicolor (Martin et al. 2004, http://genome.

jgi-psf.org/Lacbi1/Lacbi1.home.html). Because the strain 

DR 170 we used for analyses is a diploid, our results 

showed two copies.

Figure 4.—Phylogenetic analysis of Lbhsp with PAUP 4.0 

software shows its relationship to other hsp proteins. The 

following protein sequences were used for generating the 

unrooted tree. The Genbank accession number for sequences 

of the corresponding protein are indicated in parentheses. 

HSP18.2: Medicago sativa (P27880); GmHSP17.2: 

Glycine max (P04794); FhHSP17.2: Funaria hygrometrica

(CAC81966); AtHSP: Arabidopsis thaliana (NP_190209); 

SP21: Gemmata obscuriglobus (ZP_02734271); LbHSP: 

Laccaria bicolor hsp16.2 (AAM78595); MbHYP: (NP_853922); 

SpHSP16: Schizosaccharomyces pombe (SPBC3E7.02c); 

BjHSP: Bradyrhizobium japonicum (NC_00945.1); TgHSP30: 

Toxoplasma gondii (AAM62131).

Figure 5.—Temporal regulation of Lbhsp expression during 

early stages of L. bicolor x P. resinosa interactions. Northern 

analysis using RNA samples from L. bicolor subjected to 

interaction with red pine roots for 6, 12, 24, 36, 48, and 72 

hours. The bottom panel shows the picture of the gel after 

staining with SYBR stain to visualize the ribosomal RNA in 

samples that were transferred to the Hybond membrane. 

The relative intensity of these bands indicates that roughly 

the same amount of total RNA was present in each lane. 

The top panel shows the autoradiogram after hybridization to 

the Lbhsp cDNA probe. A single mRNA of ~ 600 b hybridized 

to the cDNA probe and was seen in samples that had 

interacted with plant roots for 12 to 72 hours. The sample in 

lane Fr refers to total RNA isolated from free-living fungus 

grown on MMN media on agar plates.
6

Northern analysis confi rmed that Lbhsp is expressed only 

in response to interaction with the host roots. A single 

mRNA of ~ 600 b was detected in the samples. Th e 

size of the mRNA was consistent with the nucleotide 

sequences of Lbhsp cDNA and the gene. Th e mRNA 

was not detectable in the free-living fungus grown on 

agar plates. But, within hours after interaction, it started 

appearing. We have been able to detect its presence 

as early as 4 hours after interaction as well as in the 

established ectomycorrhiza (data not shown). Th e data 

suggest that Lbhsp is essential not only for initiation and 

development of the symbiotic organ, but also for its 

maintenance.

We wanted to determine the possible role of Lbhsp in 

the ectomycorrhiza formation. Th erefore, we undertook 

further studies to determine its relationship to other 

genes expressed during early events of interaction. Since 

it has been shown in other systems that heat-shock 

proteins may interact with ras proteins, we investigated 

the possible relationship between Lbhsp and Lbras. 

Th e Lbras gene from L. bicolor is also turned on after 

interaction with plant roots as in the case of Lbhsp 

(Sundaram et al. 2001). In addition, expression of both 

followed a very similar pattern. Th e data also showed that 

Lbhsp was linked to Lbras not only at the gene expression 

level, but also at the protein level. In fi ssion yeast, 

Schizosaccharomyces pombe, Danjoh and Fujiyama showed 

that expression of a low molecular weight heat-shock 

protein, hsp16, is regulated by a ras-mediated signaling 

pathway (1999). It is possible that in the present case the 

interaction between Lbras and Lbhsp is also needed for 

the activation of the ras protein. In S. pombe, it appeared 

that hsp16 expression is regulated by the ras1-mediated 

pathway and not by the heat shock response. Similarities 

of Lbhsp to the hsp16 suggest that a similar phenomenon 

might exist in L. bicolor in its interaction with pine roots.

Analysis using the yeast two-hybrid system indicates that 

the Lbhsp protein indeed interacts with Lbras. However, 

it is not clear what precise functional role Lbhsp plays 

during signaling or interaction of L. bicolor with red pine. 

Because many hsp proteins are known to have a role as 

a chaperone, it is likely that the Lbhsp is involved in the 

protein folding leading to the activation of Lbras. In such 

a situation, the functioning of Lbras will depend upon 

simultaneous expression of Lbhsp and its interaction with 

Lbras. Th e coordinated expression of Lbhsp with Lbras in 

L. bicolor during its interaction with host roots supports 

this notion. Furthermore, in our previous studies with 

L. bicolor, we showed that Lbras may be transported 

to diff erent locations within the cell and may also be 

transported across the dolipore septum from one cell to 

the other (Sundaram et al. 2004). It has been shown in 

other systems that ras proteins are made initially in an 

inactive form and subsequently transported to diff erent 

membrane organelles or locations in the cell where 

they are transformed into active forms through proper 

refolding of the protein (Hirotsu et al. 2000). Th us, 

it is conceivable that Lbhsp may bind Lbras soon after 

its synthesis and keep it inactive until it is transported 

to a target location. Th e Lbhsp shares all the features 

of yeast hsp16 motifs, but at the sequence level is not 

highly homologous to the other hsp16 proteins. Th us, it 

is possible that Lbhsp is specifi cally involved in signaling 

events in association with Lbras. In our macroarray gene 

expression studies, we have seen coordinated expression 

of Lbhsp with Lbras (Podila et al. 2002). Taken together, 

all these results suggest that Lbras may have diff erent 

signaling circuits depending upon the developmental 

stage of the ectomycorrhiza. Further studies using GFP 

tagged Lbhsp and pull down experiments with Lbras are 

required to determine the precise dynamics of Lbhsp and 

Lbras interactions during symbiosis. Similarly, extension 

of future functional genomic studies of L. bicolor (Pandey 

et al. 2006) using Lbras and Lbhsp genes will provide 

important details of the functional role of Lbhsp and its 

importance for symbiosis.

ACKNOWLEDGMENTS

Part of the work was supported by a National Science 

Foundation grant MCB-0421326 to GKP.

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fungus Laccaria bicolor. New Phytologist. 161: 529-

538.

Voiblet, C.; Duplessis, D.; Encelot, N.; Martin, F. 

2001. Identifi cation of symbiosis-regulated gene in 

Eucalyptus globulus-Pisolithus ectomycorrhiza by 

diff erential hybridization of arrayed cDNAs. Plant 

Journal. 25: 181-191.
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Hiremath, Shiv; Lehtoma, Kirsten; Podila, Gopi K. 2009. Identifi cation of a small 

heat-shock protein associated with a ras-mediated signaling pathway in 

ectomycorrhizal symbiosis. Res. Pap. NRS-7. Newtown Square, PA: U.S. 

Department of Agriculture, Forest Service, Northern Research Station. 8 p.

Initiation, development, and establishment of a functional ectomycorrhiza involve a series 

of biochemical events mediated by a number of genes from the fungus as well as the 

host plant. We have identifi ed a heat shock protein gene from Laccaria bicolor (Lbhsp) 

that appears to play a role in these events. The size and characteristics of Lbhsp suggest 

that it belongs to the family of small heat-shock proteins described in the literature. 

Nucleotide sequencing of an almost full length cDNA indicated that the Lbhsp mRNA 

is about 611 nucleotides long and codes for a single protein of ~ 17 kDa. Isolation and 

characterization of the Lbhsp gene showed that it was made up of three exons separated 

by two small introns. Southern analysis suggested that the L. bicolor genome contains at 

least two copies of the Lbhsp gene. Temporal expression analyses revealed that the gene 

is expressed within 4 to 12 hours after interaction with red pine roots. The yeast twohybrid studies showed that the Lbhsp was closely associated with the ras gene (Lbras) 

described earlier. The data suggest that Lbhsp may play a supporting role in ras-mediated 

mycorrhizal signaling pathways during various stages of ectomycorrhizal development.

KEY WORDS: mycorrhizal fungi, heat-shock protein, symbiosis, ras-mediated
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