Physiological and Molecular Plant Pathology (1998) 52, 297–307Article No. : pp980156

Cloning and analysis of psam2, a gene from Pisum

sativum L. regulated in symbiotic arbuscular mycorrhiza

and pathogenic root–fungus interactions

F. K", F. M-L#, S. G#, V. G-P#

and P. F*"

"Max-Planck-Institut fuX r terrestrische Mikrobiologie and Laboratorium fuX r Mikrobiologie des Fachbereichs Biologie derPhilipps-Uni�ersitaX t, Karl-�on-Frisch-Straße, 35043 Marburg, Germany and #Laboratoire de Phytoparasitologie,INRA}CNRS, CMSE-INRA, BV 1540, 21034 Dijon Cedex, France

(Accepted for publication April 1998)

A differential RNA display fragment was used to isolate a full size clone from a cDNA libraryprepared from Pisum sati�um L. root RNA. The corresponding gene, psam2, belongs to a genefamily and its RNA accumulation was repressed in roots after inoculation with the arbuscularmycorrhizal fungus Glomus mosseae. No significant changes in transcript levels could be observedin the nodule symbiosis formed with Rhizobium. RNA accumulation was, however, enhanced afterinfection with two fungal root pathogens, Chalara elegans and Aphanomyces euteiches. Phosphate ornitrate fertilization had no influence on psam2 RNA expression. Sequencing revealed that theputative gene product is similar to animal secretory carrier membrane proteins. Its possibleinvolvement in changes in root exuded compounds after mycorrhizal formation or in secretionprocesses in phytopathogenic interactions is discussed. # 1998 Academic Press

INTRODUCTION

The arbuscular mycorrhiza (AM) symbiosis is a close mutualistic interaction between

soil fungi from the order Glomales and roots of most land plants [23, 25]. The

development of this symbiosis up to the morphophysiological integration is

accompanied by a number of cellular and metabolic changes [1, 14]. At least some of

these changes should be mirrored at the molecular level by the differential expression

of genes of both partners [9]. This has already been shown for several known plant

genes [15]. Non-targeted approaches like 2-D gel electrophoresis [6, 13] or differential

screening of cDNA libraries [3, 24] have been used to obtain a broader view of changes

occurring during AM interactions and to isolate new genes that are involved. We have

analysed mycorrhizal interactions using differential RNA display reverse transcription

[20]. Six plant genes showing up or down-regulation during the development of the

symbiosis were identified, and one previously unknown induced gene was investigated

in more detail [21]. Here, we present the cloning of a full size cDNA corresponding to

another unknown gene which shows repressed RNA levels during AM development.

The molecular characteristics of this gene were investigated by sequencing, Southern

*To whom correspondence should be addressed.Abbreviations used in text : dai, days after inoculation; SCAMP, secretory carrier protein.

0885–5765}98}050297­11 $30.00}0 # 1998 Academic Press

298 F. Krajinski et al.

blot analyses of genomic DNA and RNA accumulation studies in symbiotic and

pathogenic root–microbe interactions. Responses to phosphate or nitrate fertilization

were also investigated because the nutrient status of the plant changes during

mycorrhiza and nodule development [33, 36].

MATERIAL AND METHODS

cDNA cloning and analysis

A cDNA library was constructed in λZAPII (Stratagene) using 0±75 µg of poly(A)+

RNA extracted from non-mycorrhizal pea roots 21 days after germination. After

reverse transcription, double stranded cDNAs were ligated to EcoR I and Xho I

adapters and ligated into the vector following Stratagene’s recommendations. After

packaging and plating, a total number of 10( recombinants was obtained. The library

was screened with the AA01 DDRT fragment [21] labelled by PCR according to the

instructions of Gibco-BRL [22]. Positive recombinant phagemids were excised in �i�o

and used to transform XL1-Blue competent cells. For cloning of the 5« end of the

cDNA, 10( phage clones served as a template in two subsequent PCR reactions using

the nested primers psam2a.ol (CGT CTG ACT TCC TGT TCC CGT TTT C) and

psam2b.ol (GTC GAA GGG GTT AGG GTC GTA AC), as well as, in both cases, the

commercially available M13 reverse primer 18.2 (Promega). The longest PCR product

obtained was cut out from a 2% agarose gel, purified using the Geneclean system

(BIO101, Dianova) and cloned into the pT7Blue vector according to the supplier

(Novagene). Recombinant plasmids were used to transform E. coli XL1-Blue competent

cells. Several PCR and cDNA library clones were grown, plasmid DNA was isolated

on an anion exchange resin column (Genomed) and sequenced on the sequencing

automate Alf Express (Pharmacia) using the Cy54-labelled M13 universal and reverse

primers 18.1 and 18.2 (Biological Detection Systems) following the method of Sanger

et al. [32]. The sequence appears in the EMBL}Genebank under the accession number

AF018093. Sequence comparisons and protein analyses were carried out with the DNA

Star programs Align, Megalign and Protean using data from the EMBL}GeneBank.

Genomic Southern blots

Genomic DNA was extracted from leaves of pea plants 21 days after germination [5],

10 µg were digested with EcoR I and BamH I, separated by electrophoresis on a 1%

agarose gel and transferred by capillarity onto nylon membranes (Hybond N,

Amersham). DNA fragments were labelled as described for the library screening

and hybridized to the genomic DNA at 65 °C [31]. The probe for the 3« un-

translated region was amplified by PCR using the primer psam2c.ol

(GTG AAG GCA GTA TGA ATG TC) and the M13 universal primer 18.1

(Promega).

Plant growth and inoculation

Pea plants (Pisum sati�um L. cv Frisson) were grown in the presence or absence of the

AM fungal isolate Glomus mosseae (Nicol and Gerd) Gerd. and Trappe (BEG 12) as

described previously [7]. For nodule development, a suspension of Rhizobium

leguminosarum b.v. �iciae (Isolate 1007, INRA Dijon) was directly added to the growth

Cloning and analysis of psam2 299

substrate according to the method of Sagan et al. [30]. For interactions with a

pathogen, mycelium of Chalara elegans Nag. Raj Kendrick was taken from plates,

resuspended carefully in water and incubated for 10 min with roots of 7-day-old pea

seedlings before planting [38]. For comparison with a different pathogen, Aphanomyces

euteiches Drechs. was inoculated by watering 7-day-old pea seedlings with a zoospore

suspension after planting [34]. Roots were harvested for RNA analysis at 6 or 15 days

after inoculation (dai) for the symbiotic micro-organisms, and 1 or 15 dai for the

pathogens. Infections of the different micro-organisms were observed by light

microscopy after staining with trypan blue [27]. Parameters of mycorrhizal colonization

were determined on root samples according to Trouvelot et al. [40]. In the case of the

other micro-organisms, 30 randomly chosen root fragments of 1 cm were analysed by

light microscopy. In order to analyse the effect of P or N, plants were grown for 3 weeks

and fertilized every 3 days with Long Ashton solution (2 ml per 100 ml substrate) with

and}or without the addition of phosphate or nitrate [18].

Northern blot analyses

Total RNA was isolated from pea roots according to the method of Franken and

Gna$ dinger [8], and 5 µg of each samplewere blotted after denaturing gel electrophoresis

onto nylon membranes (Hybond N, Amersham) by capillary transfer [31]. Blots were

hybridized at 65 °C with the psam2 cDNA and washed under standard conditions [31].

After autoradiography, they were stripped at 80 °C for 30 min in 0±1% SDS and

subsequently hybridized with an rRNA probe [10]. Probes were labelled as indicated

for the screening of the library. Northern blots were repeated three times for

mycorrhiza and twice for other infections and for the fertilization experiments. The

autoradiographs were scanned using the Ultrascan XL (LKB, Sweden).

RESULTS

Structure of psam2

A total of 180000 plaques of a cDNA library prepared with RNA extracted from non-

mycorrhizal roots of Pisum sati�um were screened with the AA01 DDRT fragment

described by Martin-Laurent et al. [21]. Four positive clones were obtained with

different lengths. Sequencing revealed that they all belonged to the same gene, but

none of them were of full size. A PCR experiment was therefore carried out using

phages of the library as template. The combination of the data from the cDNA clones

and from the PCR fragment revealed a sequence (Fig. 1) which corresponds in length

to the transcript obtained in northern blots (1400 bp). This sequence carried an ORF

which encodes a gene product (Fig. 2a) showing similarity to secretory carrier

membrane proteins in animals [2, 43]. Although this similarity is low, it is distributed

over the whole amino acid sequence (Fig. 2b).

Genomic organisation of psam2

Southern blot analysis was conducted using genomic DNA digested with EcoR I or

BamH I and hybridized with the whole cDNA psam2 clone as probe (nt 208–nt 1400 in

Fig. 1). Each restriction enzyme gave three or four strong bands, respectively, as well

as several weak bands after autoradiography (Fig. 3, lane 1 and 2) indicating the

300 F. Krajinski et al.

F. 1. Sequence of psam2. The psam2 cDNA sequence obtained from the phage library cloneand the PCR experiment is shown. Putative start and stop codons, as well as possible adenylationsignals are boxed and the oligonucleotides which were used for amplification of the 5« or the 3«region are marked with arrows (psam2a.ol : M N, psam2b.ol : MN and psam2c.ol : –––N). The5« end of the phage library cDNA clone is indicated with a vertical arrow.

Cloning and analysis of psam2 301

300

200

100

100

23%

PSAM2

SC

AM

P (

R.n

.)

200

300

200

100

100

30%

PSAM2

SC

AM

P (

C.e

.)

200

300

200

100

100

37%

SCAMP (C.e.)

SC

AM

P (

R.n

.)

200 300

(b)

F. 2. Comparison of PSAM2 with SCAMP from different animals. (a) The amino acidsequence encoded by psam2 (line 1) is aligned to those of secretory carrier membrane proteins fromCaenorhabditis elegans (line 2; AF003739) and Rattus nor�egicus (line 3, L22079). Residues which areidentical are indicated by stars and those with conservative exchanges are underlined in theSCAMP sequences. (b) The same amino acid sequences were used for a dot plot analysis.Similarities are given below the plots.

302 F. Krajinski et al.

F. 3. Genomic Southern blot. Genomic DNA of Pisum sati�um L. was digested with EcoR I(lane 1 and 3) or BamH I (lane 2 and 4), separated by gel electrophoresis, transferred andhybridized to the psam2 cDNA clone (lane 1 and 2) or to the 3« untranslated region (lane 3 and4). The sizes of the marker DNA (lane M) are indicated.

T 1Mycorrhization parameters of Glomus mosseae in roots of Pisum sativum L.

Days after inoculation

Colonization 6 15

F%* 69±1 (­}® 20)§ 85±2 (­}® 7±5)M%† 4±5 (­}® 4±1) 25±0 (­}® 8±5)A%‡ 0±0 18±3 (­}® 5±8)

*Frequency of infected root fragments.†Colonization intensity of root cortex.‡Arbuscule abundance within the root system.§Standard deviations from three independent experiments.

Cloning and analysis of psam2 303

F. 4. Northern blot analyses. In order to study the accumulation of psam2 transcripts insymbiotic plant-microbe interactions (a), RNA was extracted 6 dai and 15 dai from control roots(C), mycorrhiza (M) and roots infected with R. leguminosarum (R). (b) For the pathogenic fungiA. euteiches (Ap) or C. elegans (Ch), RNA was extracted 1 dai and 15 dai. Effect of nutrients wereanalysed (c) with RNA from roots four weeks after planting where pots were fertilized with thecomplete Long Ashton solution (lane 1), a solution without P (lane 2) or without N (lane 3) andwithout both N and P (lane 4). In all cases, 5 µg of these RNA extractions were hybridizedsubsequently in northern blots with the psam2 cDNA clone and a rRNA gene as probes.Autoradiography was done in all cases over night except for the pathogens 1 dai, where it wascarried out only for 2 h.

presence of a gene family in pea. In order to detect the member of this family

corresponding to psam2, the untranslated 3« region of the gene (nt 1081–nt 1400 in

Fig. 1) was used as probe with the same genomic DNA. Only one clear band could be

detected after hybridization with this more specific probe (Fig. 3, lane 3 and 4). These

results indicate that psam2 most probably belongs to a multigene family in pea, with at

least two members. Only one member of this gene family seems to be expressed in roots,

because all clones which were obtained from the library carried the the same sequence.

Interestingly, in humans, SCAMPs are also encoded by a gene family [35].

304 F. Krajinski et al.

RNA accumulation of psam2

For the analysis of psam2 RNA accumulation in different root–microbe interactions,

comparisons were made between pea plants inoculated with G. mosseae, R. leguminosarum,

A. euteiches and C. elegans. Infection development was checked microscopically in all

cases. Values for mycorrhiza development are given in Table 1. At the early time point

(6 dai), about 70% of the mycorrhizal root system showed infection structures (F%),

but colonization within roots was very low (M%) and no arbuscules (A%) could be

observed. When the symbiosis was fully established (15 dai), about 1}4 of the root

system was colonized by mycelium of G. mosseae and arbuscules had developed. In the

case of infection by Rhizobium, 6 dai no obvious nodules had formed, but infection

threads could be observed. About 20 root nodules per plant had developed by the later

time point (15 dai). In interactions with the fungal pathogens, the mycelium of C.

elegans colonized the root surface and single hyphae were already infecting the roots

1 dai. Unfortunately, hyphae of A. euteiches could not be observed at this time point, as

they did not stain with trypan blue nor with two other dyes (chlorazole black E, fuchsin

red). However, 15 dai roots were heavily colonized by hyphae of C. elegans or full of

oospores of A. euteiches and plants showed disease symptoms (reduced plant growth,

necrotic root regions). Control roots were free of any fungal or bacterial structures at

both time points.

Five micrograms of RNA extracted from control and infected roots were separated

by gel electrophoresis, transferred onto a nylon membrane, and sequentially hybridized

to the psam2 cDNA clone and a rRNA probe (Fig. 4a and b). The autoradiograms were

analysed by image analysis and the values derived from the hybridization with the

psam2 cDNA were normalized by the data from the rRNA probe and expressed as

percentages of the corresponding controls (Table 2). The results revealed a slight

T 2Relati�e changes of psam2 RNA le�els*,†

Days after inoculation

Treatment 1 6 15

­}® Glomus mosseae n.m.‡ 80% 5%­}® Rhizobium leguminosarum n.m. 78% 63%­}® Chalara elegans 485% n.m. 51%­}® Aphanomyces euteiches 982% n.m. 63%

*Average values (deviations were below 10%).†Controls were set as 100%.‡Not measured.

decrease in psam2 RNA accumulation during nodule development 6 and 15 dai. A clear

decrease in the amount of psam2 transcripts could be detected at the later time point

(15 dai) after G. mosseae inoculation, when the mycorrhizal symbiosis was fully

developed. Analysis of infection with the pathogenic fungi showed that psam2 transcript

accumulation was strongly enhanced 1 dai, but this difference decreased at the later

Cloning and analysis of psam2 305

time point. No change in psam2 RNA accumulation was observed in response to

phosphate and nitrate fertilization (Fig. 4c).

DISCUSSION

In order to understand the molecular basis of arbuscular mycorrhiza (AM), research

in the last few years has been aimed at analysing gene expression during plant–fungal

interactions in the symbiosis. Up to now, most investigations have been focused on

genes known from other plant–microbe interactions, such as those involved in defence

reactions [15] or in nodule development [12, 29, 41]. However, several new genes have

been cloned recently using non-targeted approaches [3, 21, 24], but their regulation

during interactions with other micro-organisms is not known. In the present

investigation, we have shown that RNA accumulation of the gene psam2 from pea is

repressed at late stages of arbuscular mycorrhiza development, but induced in early

interactions with plant pathogenic fungi, whilst only a slight change in expression of

this gene occurs during nodule development. It also does not respond to phosphate

nutrition, contrary to what was observed for a mycorrhiza-repressed gene in Medicago

truncatula [3]. Psam2 could belong to a group of genes related to plant defence or

pathogenesis which are repressed in mycorrhiza, like those encoding glucanases and

chitinases [19] or enzymes involved in phytoalexin production [8, 17, 42]. A gene

similar to psam2 has not been reported up to now in plants, and may therefore present

a member of a new class of pathogenesis related (PR) genes.

The structure of the psam2 encoded peptide revealed from the deduced amino acid

sequence has a low similarity to a family of secretory carrier membrane proteins

(SCAMP) in animals. This similarity is, however, distributed over the whole sequence

and the secondary structures indicated from hydrophobicity plots or predicitons for

alpha helices also seem to be fairly similar (data not shown). Therefore, it can be

speculated that the PSAM2 protein has a function related to that of SCAMPs. SCAM

proteins are markers of the general cell surface recycling system and play a role in

secretion processes [2, 35]. During infection with the C. elegans and A. euteiches, PSAM2

could therefore eventually be involved in the secretion of extracellular PR proteins or

antifungal phenolic compounds which have often been reported to accumulate in

plants after pathogen attack [4, 26]. Transient increases of RNA at the beginning of

plant’s response to a pathogen or an elicitor has been observed for different genes in

other systems [11, 37]. It has been shown that the composition of root exudates changes

during mycorrhiza development, but the observed decreases in sugars and amino acids

[16, 28, 39] or certain phenolic compounds [8] could also be observed after improved

phosphate nutrition and seemed to be a membrane-mediated process [16]. More

studies are necessary to demonstrate clearly that the psam2 gene product is involved in

secretory processes and to relate its activity to substances which are less exuded in the

mycorrhizal symbiosis, but not affected by phosphate fertilization.

We wish to thank Muriel Sagan for providing us with the R. leguminosarum strain and

Soren Rosendahl for the A. euteiches isolate. F. Krajinski was supported by PROCOPE

(Contract 93134) and F. Martin-Laurent by a Conseil Regional de Bourgogne}INRA

grant (Contract 5315B) and PROCOPE (Contract 93134).

306 F. Krajinski et al.

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