Differential expression of fungal genes at preinfection and mycorrhiza ...

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Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones Blackwell Publishing Ltd

Marianna Zaretsky1, Yaron Sitrit2, David Mills2, Nurit Roth-Bejerano1 and Varda Kagan-Zur1 1

Department of Life Sciences and 2The Jacob Blaustein Institutes for Desert Research, Ben Gurion University, POB 653, Beer Sheva, Israel

Summary Author for correspondence: Varda Kagan-Zur Tel: +972 8 6461974 Fax: +972 8 6472984 Email: [email protected] Received: 5 February 2006 Accepted: 20 April 2006

• Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. • Four fungus–hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA–AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. • Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. • Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza. Key words: cDNA–AFLP (amplified fragment length polymorphism), reverse Northern, mycorrhizal type determination, gene expression, Terfezia boudieri. New Phytologist (2006) 171: 837–846 © The Authors (2006). Journal compilation © New Phytologist (2006) doi: 10.1111/j.1469-8137.2006.01791.x

Introduction Ectomycorrhizas are mutualistic symbiotic associations between plant roots and compatible fungi. The development and maintenance of a functioning ectomycorrhiza entrains extensive changes in the genes that control signaling and metabolic pathways. The formation of the ectomycorrhizal root takes place in four stages – preinfection, colonization, differentiation, and functioning (Martin et al., 1997). A complex series of molecular mechanisms is activated at the earliest or preinfection stage that precedes actual contact between plant and fungus (Menotta et al., 2004). Preinfection plant genes code for proteins implicated in signal perception, signal transmission, stress responses, metabolism and growth (Krüger et al., 2004). Fungal presymbiotic genes are likely to

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be involved in cellular organelle dynamics, cell wall construction, fungal metabolism and cellular detoxification processes, cellular signaling, cell-cycle completion, patterning and protein transport across the cell wall, as well as transcriptional regulation of development (Podila et al., 2002; Menotta et al., 2004). Duplessis et al. (2005) have shown that, at the earliest stages of ectomycorrhizal development, differentially expressed fungal genes include cell wall symbiosis-regulated proteins, as well as hydrophobins and mannoproteins. Changes in gene expression have been observed at the levels of transcripts (Voiblet et al., 2001; Podila et al., 2002; Polidori et al., 2002) and proteins (Burgess et al., 1995; Tarkka et al., 1998). Later ectomycorrhizal stages – formation and differentiation – have been shown to involve up- or downregulation of existing functional pathways in both partners, encompassing

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genes that promote cell growth, biosynthesis, differentiation and signaling, synthesis of cell surface and extracellular matrices, and primary metabolism (Nehls et al., 1998; Nehls et al., 2001; Voiblet et al., 2001; Balasubramanian et al., 2002; Peter et al., 2003; Tagu et al., 2003; Sundaram et al., 2004). Mid- and late-transcriptionally responsive fungal genes code for the enzymes of glycolysis, the tricarboxylic acid cycle, and amino acid biosynthesis, as well as for protein synthesis, hormone metabolism, and signal transduction components; also included in this category are mycelium-specific genes of unknown function (Duplessis et al., 2005). However, no ectomycorrhiza-specific genes have been identified (Voiblet et al., 2001; Duplessis et al., 2005). Le Quéré et al. (2004) demonstrated that, in two closely related strains of Paxillus involutus, varying host specificity was associated with changes in the patterns of plant-induced gene expression. In a previous study of mycorrhizas formed under limiting phosphate levels between Cistus incanus hairy root clones and Terfezia boudieri isolates, we found that only one plant root clone was capable of forming endomycorrhizas with only two of the five Terfezia isolates tested (Zaretsky et al., 2006). All other plant clone–fungal isolate combinations resulted, under the same conditions, in a Terfezia-type ectomycorrhiza (Zaretsky et al., 2006). Little is known about fungal differential gene expression during preinfection stages or the pathways that lead to ecto- rather than endomycorrhizal formation. A study was thus conducted on four Terfezia isolate–Cistus hairy root clone combinations cultivated under two sets of conditions. In the first set, plant clones and fungal isolates were separated by a cellophane sheet; the second set allowed physical contact (referred to as preinfection and mycorrhiza stages, respectively). Under low phosphate levels, only one combination engendered endomycorrhizal formation (Zaretsky et al., 2006), all the other combinations resulting solely in ectomycorrhizas (Zaretsky et al., 2006). cDNA– AFLP analysis was used to isolate fungal genes that were differentially regulated under the preinfection stage in two different fungal isolates. A macroarray was then constructed with the obtained cDNA fragments, to investigate in more detail gene expression under preinfection and mycorrhizal stages. Our findings lead us to define three groups of genes involved in mycorrhiza establishment: transiently expressed genes (expressed only under preinfection conditions); genes expressed solely under mycorrhiza-enabling conditions; and genes exhibiting altered expression patterns between the two sets of conditions. We also found that different combinations of genes were involved in ecto- vs endomycorrhizal formation.

Materials and Methods Fungal and plant material The Terfezia boudieri Chatin isolates used in this study were routinely grown on Fontana medium (Bonfante & Fontana,

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Table 1 Phenotypic characteristics of Terfezia boudieri isolates and Cistus incanus hairy root clones, and the list of combinations describing mycorrhiza type formed for each pair as used in the study Fungal isolates and plant clone characteristics: Fungal isolate/ plant clone

Growth rate

Isolate 42a Isolate 27 Clone M2 Clone W51

Fast Fast Fast Fast

Sensitivity to IAA

IAA excretion Low High

High Low

Combinations studied: Fungal isolate

Plant clone

Mycorrhiza type formed

Isolate 42a Isolate 42a Isolate 27 Isolate 27

M2 W51 M2 W51

Endomycorrhiza Ectomycorrhiza Ectomycorrhiza Ectomycorrhiza

Data from Zaretsky et al. (2006).

1973) pH 6, solidified with 1.5% Difco bacto agar. The Cistus incanus L. hairy roots were obtained as described by Zaretsky et al. (2006), and routinely subcultured every 3 wk on N5 medium [MS medium (Murashige & Skoog, 1962) with 20% the amount of nitrates] solidified with 0.2% phytagel. Table 1 describes fungal isolates and plant clones used in this study. Growth conditions For this set of experiments, Cistus hairy root clones and T. boudieri isolates were grown as described below on medium M (Bécard & Fortin, 1988) at pH 5.5, solidified with 0.2% phytagel modified with respect to phosphorus concentration and containing 0.48 mg l−1 KH2PO4 (low P, medium M). Free-living T. boudieri isolates For the initial AFLP experiments, isolates were grown for 4 wk on an M-medium plate covered with a piece of cellophane (King, Yuyao, China). Before use, the cellophane sheets were boiled for 10 min in H2O containing 1 mM EDTA, washed twice in distilled water, and autoclaved. The T. boudieri isolate was then placed on the cellophane and kept in the dark for 4 wk. Fungal isolates were then collected, frozen in liquid N2, and stored at −80°C until RNA extraction. Separated cultivation of roots and T. boudieri on the same plates Three transformed plant clone root tips were transferred to an M-medium plate and covered with a piece of cellophane prepared as described above. The plates were kept in the dark for 4 wk. Fungal isolates and plant root clones were then collected separately, frozen in liquid N2, and stored at −80°C until RNA extraction.

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Mycorrhized roots To obtain mycorrhized roots, plant clones were grown in tubes for 2.5 wk before inoculation with a cube of Fontana (Bonfante & Fontana, 1973) agar medium containing the T. boudieri isolate. After inoculation, tubes were maintained in a growth room at 25°C in the dark for an additional 4 wk. The mycorrhized roots were then collected, frozen in liquid N2 and stored at −80°C until RNA extraction. RNA extraction RNA was extracted from fungal isolates according to the protocol for the RNAgents Total RNA Isolation System (Promega, Madison, WI, USA). RNA was extracted from nonmycorrhizal and mycorrhized plant root clones using the protocol described by Wilkins & Smart (1996) with several modifications. Briefly, plant tissue was homogenized in liquid N2 using a mortar and pestle, and transferred to tubes containing homogenization buffer (10 mM Tris–HCl pH 8.5, 100 mM LiCl, 5 mM EDTA pH 8.0, 100 mM NaCl, 1.0% sodium dodecyl sulfate, SDS). The tubes were centrifuged and the aqueous phase was collected and extracted once with phenol : chloroform : isoamyl alcohol (25 : 24 : 1), then centrifuged and extracted again with chloroform : isoamyl alcohol (24 : 1). RNA was precipitated with NaAc and ethanol and centrifuged, and the pellet was resuspended in diethylpyrocarbonate-treated H2O. Any potential DNA contamination in RNA samples was removed by DNAse (Promega) treatment. cDNA–AFLP analysis cDNA–AFLP analysis was conducted on fungal isolates harvested under free-living and preinfection conditions. First-strand cDNA synthesis was performed on 5 µg total RNA using Superscript II reverse transcriptase (Invitrogen, Paisley, UK) as indicated by the manufacturer. Second-strand cDNA synthesis was carried out according to Ausubel et al. (2003). The resulting double-stranded cDNA was phenol-extracted, ethanol-precipitated, and resuspended in a final volume of 40 µl ddH2O (Kashkush et al., 2002). Formation of the expected products between 100 and 3000 bp was verified on 1.5% agarose gel (Hispanagar, Burgos, Spain). Three cDNA samples of the required quality from three different RNA isolations were combined and subjected to a standard AFLP analysis (Vos et al., 1995) using MseI and EcoRI enzymes. Bands showing differential display were excised using a sharp, clean razor blade. DNA-containing gel pieces were placed in 1.5 ml microcentrifuge tubes with 50 µl H2O and incubated at 37°C overnight. Cloning of excised AFLP fragments Each cDNA fragment was reamplified using the corresponding primers employed at the selective AFLP amplification. PCR cycles were: 3 min at 94°C; 34 cycles of 30 s at 94°C, 1 min at 56°C and 1 min at 72°C; followed by 10 min at 72°C. The size of each band was determined on 2% agarose gel and compared with the size

estimated from the AFLP gel. To increase specificity, annealing temperatures between 56 and 62°C were used until amplification of a single band was observed. PCR products were cloned into pTZ57R/T (MBI Fermentas, Vilnius, Lithuania) or pGEM (Promega) vectors and transformed into Escherichia coli XL1-blue in accordance with the manufacturer’s instructions. Positive colonies were grown in Luria broth (Miller, 1972) overnight, and plasmids were extracted by the alkaline method (Sambrook & Russell, 2001) and sequenced. A BLAST search for nucleotide and protein homologies was made using the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/blast) and formed the basis for functional annotation. Sequences with an E value ≤ 1.0e −5 were considered to identify known genes or to have partial similarity to known genes; others were classified as no match. Cluster analysis was performed for all differentially expressed genes using CLUSTALW software for multiple alignment (http://www.ebi.ac.uk/clustalw/index.html). Macroarray Macroarray was used to investigate in more detail gene expression under preinfection and mycorrhizal conditions in various plant root–fungal isolate combinations. Each cDNA fragment was reamplified as described above and purified on Sephadex™ G-50 Fine columns (Amersham Biosciences, London, UK) prepared as described by Sambrook & Russell (2001). The cDNA fragments (100 ng) were denatured and dot-blotted onto Hybond-N+ nylon membrane (Amersham Biosciences) according to the protocol described by Ausubel et al. (2003) using Bio-Dot® microfiltration apparatus (Bio-Rad, Hercules, CA, USA). Each cDNA fragment was spotted twice on the membrane, and three membrane duplicates were prepared. Each reverse Northern dot-blot experiment was repeated twice on different membrane replicas using six independent RNA extractions and cDNA preparations (in groups of three). The RNA for hybridization was extracted from each of the fungal isolates (42a and 27) grown under preinfection conditions with plant clones (M2 and W51), and also from the mycorrhized roots (each plant clone with each fungal isolate, in all eight probes). Total RNA (5 µg) was labeled with [α-32P]dCTP (Amersham Biosciences) during reverse transcription with the enzyme Superscript II (Invitrogen). Unincorporated nucleotides were removed using ProbeQuant™ G-50 microcolumns (Amersham Biosciences, Piscataway, NJ, USA). The membranes were prehybridized at 65°C for at least 3 h in hybridization buffer [5 × standard saline citrate (SSC) solution, 5 × Denhardt’s solution, 0.5% SDS, 100 ng µl−1 denatured salmon sperm DNA (Ausubel et al., 2003)]. Denatured probe was then added and overnight hybridization was carried out. Membranes were then washed successively at 65°C for 2 × 5 min in 2 × SSC containing 0.1% SDS in the hybridization tubes, transferred to plastic boxes to allow better removal of

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unspecific bound probe, washed 2 × 10 min in 1 × SSC containing 0.1% SDS, and then 3 × 5 min in 0.5 × SSC containing 0.1% SDS. Membranes were exposed to X-ray film (Kodak X-OMAT AR, Sigma, Israel) at −80°C for 24 h for a short exposure and for 1 wk in longer exposures. Each membrane was hybridized with RNA isolated from plant clones grown under preinfection conditions with each of the fungal isolates. No cross-reaction with fungal genes was detected. However, some plant genes expressed under mycorrhizal conditions, but not during the preinfection stage, may still cross-react. cDNA fragments that showed differential display in any of the comparisons were deposited in dbEST at the National Center for Biotechnology Information (NCBI accession numbers DT639222, DT639223, DV205746–DV205817).

Results Overall distribution of sequence categories cDNA–AFLP was performed with RNA extracted from two fungal isolates (42a and 27) cultivated alone (free-living) or in the presence of plant clones M2 or W51 (preinfection). In the latter case,

fungal isolates were separated from plant roots by a piece of cellophane. Amplification resulted in approx. 300 cDNA fragments exhibiting differential expression, of which 279 were cloned and subjected to reverse Northern dot-blot analysis. The reverse Northern analysis revealed that 157 putative fungal gene representatives gave no signal at all. Of those, 19 were originally isolated from individually grown fungal isolates and hence were not expected to be expressed in fungus–root combinations. An additional 48 cDNA fragments, almost half of which exhibited homology to rRNA genes, were expressed in all combinations under all conditions (data not shown). The remaining 74 cDNA fragments showed differential expression in one or more of the combinations grown under preinfection and/or mycorrhized conditions (Fig. S1 in Supplementary Material). Cluster analysis performed on these sequences revealed five groups of identical cDNA fragments comprising 16 sequences. One cDNA fragment from each group was left while others were dropped, resulting in 63 cDNA fragments. These genes were grouped into several categories according to the type of expression (Fig. 1a): 59% of the 63 reporters examined yielded signal only when testing

Fig. 1 Fungal gene expression under preinfection and mycorrhizal growth conditions as revealed by reverse Northern analysis. (a) Distribution of cloned fungal cDNA fragments over experimental condition types. (b) Distribution of differentially expressed genes over functional categories at the preinfection and mycorrhiza stages. Functional categories: 1, metabolism; 2, vesicular trafficking, secretion and protein sorting; 3, cell wall; 4, gene expression; 5, protein synthesis and processing; 6, membrane transport; 7, signal transduction and post-translational regulation; 8 unknown function; 9, no match. The number of genes allocated to each functional category is indicated.



Research Table 2 Terfezia boudieri genes expressed exclusively under preinfection conditions, as examined by reverse Northern analysis Fragment no.

GenBank accn no.

42a+M2 27+M2

42a+W51

27+W51 Homology

Function*

E

Size (bp)

+ + + + + + + + + + + + + + + + + + +

Amino transferase Endosomal Vps protein complex subunit Dipeptidyl-peptidase IV No match No match No match No match No match No match No match No match No match No match No match No match No match No match No match No match

1 2 7 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

8e−92 2e−13 2e−56 – – – – – – – – – – – – – – – –

811 243 621 128 332 175 245 249 98 51 242 72 240 133 213 116 239 362 446

Fungal gene expression associated with plant clone W51 3 DT639222 + +

No match

9

–

200

Fungal gene expression associated with ectomycorrhiza formation 13 DV205747 + + + 246-2 DV205798 + + + 229 DV205792 + + + 245 DV205796 + + + 181b-2 DV205774 + + +

Glycogen branching enzyme No match No match No match No match

1 9 9 9 9

2e−19 – – – –

141 290 386 81 68

Fungal gene expressed in all combinations of fungal isolates and plant root clones 49 DV205760 + + + + Cytochrome c oxidase subunit I 121 DV205772 + + + + Small subunit rRNA 214 DV205783 + + + + Small subunit rRNA 91 DV205766 + + + + Plasma membrane ATPase 29-2 DV205756 + + + + WD40 domain 34 DV205758 + + + + Hypothetical protein 39-1 DV205759 + + + + Hypothetical protein 12-2 DV205746 + + + + No match 291 DV205813 + + + + No match

1 5 5 6 7 8 8 9 9

5e−12 3e−35 6e−18 2e−60 7e−34 1e−23 1e−5 – –

396 120 111 450 318 428 154 254 208

Fungal genes expressed in only one combination of a fungal isolate and a plant clone 219b-1 DV205786 + Endopolygalacturonase 1 (pectinase) 19 DV205751 + No match

3 9

5e−7 –

364 79

Fungal gene expression associated with fungal isolate 27 30-2 DV205757 + 243-2 DV205795 + 234 DV205793 + 97-2 DV205769 + 28-2 DV205754 + 259 DV205799 + 207 DV205781 + 270-2 DV205802 + 286 DV205809 + 261 DV205800 + 226-2 DV205790 + 11-3 DT639223 + 97-1 DV205768 + 227 DV205791 + 271-1 DV205803 + 271-2 DV205804 + 274-1 DV205805 + 22 DV205752 + 215a DV205784 +

*Functional category of expressed genes: 1, metabolism; 2, vesicular trafficking, secretion and protein sorting; 3, cell wall; 4, gene expression; 5, protein synthesis and processing; 6, membrane transport; 7, signal transduction and post-translational regulation; 8, unknown function; 9, no match.

preinfection tissues (Table 2); 14% only when testing the mycorrhizal tissues (Table 3); and 27% of the reporters gave a signal in both tissues tested (Table 4). Disregarding fragments with no match to GenBank sequences, metabolism-related genes constitute the largest group under the preinfection condition, while genes involved in protein synthesis are the most numerous when both stages are considered together (Fig. 1b). At all mycorrhizal stages tested, fragments showing no homology to any published sequence constitute the largest group.

Preinfection unique genes The 37 fungal genes expressed by the fungal isolates under cellophane-separated growth conditions are listed in Table 2. In this category, the single largest group is that of fungal isolate 27-associated genes. This group consists of 19 cDNA fragments belonging to different functional classes expressed by fungal isolate 27 in the presence of both M2 and the W51 plant clone. Most of the cDNA fragments showed no homology,


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842 Research Table 3 Terfezia boudieri genes expressed exclusively under mycorrhitic conditions, as examined by reverse Northern analysis Fragment no.

GenBank accn no.

42a+M2

27+M2

42a+W51

27+W51

Homology

Function*

E

Size (bp)

+ +

No match No match

9 9

– –

141 262

+

+

No match

9

–

126

Fungal gene expression associated with ectomycorrhiza formation 205 DV205779 + + 269 DV205801 + + 25 DV205753 + +

+ + +

No match No match No match

9 9 9

– – –

192 334 175

Fungal gene expressed in all combinations of fungal isolates and plant root clones 63-2 DV205764 + + + +

No match

9

–

167

Fungal genes expressed in only one combination of a fungal isolate and a plant clone 183-1 DV205775 +

No match

9

–

118

Fungal gene expression associated with the fungal isolate 27 54 DV205761 + 61 DV205763 + Fungal gene expression associated with plant clone W51 102 DV205771

*Functional category of expressed genes: 1, metabolism; 2, vesicular trafficking, secretion and protein sorting; 3, cell wall; 4, gene expression; 5, protein synthesis and processing; 6, membrane transport; 7, signal transduction and post-translational regulation; 8, unknown function; 9, no match.

while others belonged to genes involved in cell metabolism (amino transferase), signal transduction (dipeptidyl-peptidase IV) and cellular transport. In addition, a single gene was found to be plant clone W51-specific and showed no homology to any known gene. Five genes proved to be ectomycorrhiza-associated, as they were expressed by all fungal isolate–plant clone couples except the endomycorrhiza-forming combination (42a+M2). Only one fragment showed homology (to a glycogen-branching enzyme that may be involved in metabolism), while the rest showed no homology to any GenBank sequence. Nine genes were uniformly expressed by all fungus–root combinations; of these, two showed homology to predicted proteins, while others were similar to genes involved in different cellular processes, such as metabolism (cytochrome c oxidase), protein synthesis (rRNA genes) and membrane transport (ATPase). Two additional cDNA fragments showed no homology. Pectinase and a cDNA fragment showing no homology were expressed only in a single fungal isolate–plant clone combination (27+M2) and were classified as a single combination expression group. Mycorrhiza-unique genes Nine putative fungal gene representatives expressed only under mycorrhiza-favoring conditions are listed in Table 3. Neither of these cDNA fragments showed homology to the databases. Two fungal isolate 27-associated genes were identified; three other cDNA fragments were ectomycorrhiza-specific and were expressed by all ectomycorrhiza-forming combinations; only one gene was found to be expressed in all four fungus–plant clone combinations under these conditions.


Genes expressed under both sets of growth conditions Seventeen cDNA fragments were expressed under both preinfection and mycorrhiza-favoring conditions (Table 4). Eight putative fungal genes were expressed uniformly by all combinations under preinfection conditions, but had variable expression patterns under mycorrhiza-favoring conditions. Four genes were found to be specific for the plant clone M2, and were expressed by both fungal isolates when grown with this plant clone in physical contact. One fungal isolate 42a-specific gene probably involved in protein synthesis was also identified under mycorrhiza-favoring conditions. Four genes were endomycorrhiza-specific (the M2+42a combination) when grown under mycorrhiza-favoring conditions. Of these, one showed high similarity to rRNA genes; the remaining three lacked homologs and were ectomycorrhizaspecific (the M2+27, W51+42a and W51+27 combinations) under preinfection conditions.

Discussion The aim of this study was to compare fungal gene expression during preinfection and mycorrhizal growth conditions leading to ecto- or endomycorrhiza formation. As two different T. boudieri isolates were grown in the presence of the two different C. incanus hairy root clones, our results also allowed us to investigate possible differences in gene expression in the same fungal isolate in response to different plant clones, on the one hand; and the influence of a particular plant root clone on gene expression in different fungal isolates, on the other hand. Martin et al. (1997) maintain that ectomycorrhiza formation can be broadly divided into four stages – preinfection, initiation, differentiation, and functioning. In our in vitro



Table 4 Terfezia boudieri genes expressed under both preinfection and mycorrhiza stage Cellophane-separated conditions Fragment no.

GenBank accession no.

290 294-2 295-1 212 136-2 206 223 14 101 226-1 55-2 287 293-1 29-1 246-1 221 242

DV205812 DV205816 DV205817 DV205782 DV205773 DV205780 DV205788 DV205748 DV205770 DV205789 DV205762 DV205810 DV205814 DV205755 DV205797 DV205787 DV205794

Mycorrhized conditions

42a+M2 27+M2

42a+W51

27+W51

+ +

+ +

+

+

+ + + + +

+ + + + + +

+

+ + + + + + + + + +

+ + + + + + + + + +

+

+ + +

+

+ + + + +

+ + + +

42a+M2 27+M2

+ + + + + + +

+ + +

42a+W51

+ + + + + + + +

27+W51

Homology

Function* E

+ +

Cytochrome-C oxidase Vacuolar sorting-associated protein vps13 Small subunit rRNA gene Small subunit rRNA 18s rRNA ABC transporter ras-2 protein No match No match No match No match No match No match No match No match No match No match

1 2 5 5 5 6 7 9 9 9 9 9 9 9 9 9 9

+ +

+ +

+ + +

+

+

+

+

+ + +

2e−4 9e−7 2e−52 1e−35 1e−20 2e−35 3e−8 – – – – – – – – – –

Size (bp) 191 169 167 146 70 362 454 200 93 89 282 81 219 317 81 51 280

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*Functional category of the expressed genes: 1, metabolism; 2, vesicular trafficking, secretion and protein sorting; 3, cell wall; 4, gene expression; 5, protein synthesis and processing; 6, membrane transport; 7, signal transduction and post-translational regulation; 8, unknown function; 9, no match.

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model system, the first three stages usually occur during the first month of physical contact between the two partners (data not shown). We chose to concentrate on late stages of mycorrhiza formation, and therefore examined fungal gene expression after 1 month of joint growth. Genes that were expressed consistently or not at all, at both mycorrhizal stages studied here, were not taken into account in this work as they appear to have played no role in ecto- or endomycorrhizal formation and maintenance at the time points examined. However, some of these genes may be differentially expressed earlier or later in the mycorrhizal process. As the purpose and essence of the mycorrhiza is to mediate coordinated nutrient exchange (Sundaram et al., 2004), we may expect that genes involved in signaling, biosynthesis, metabolism and vesicular traffic (Voiblet et al., 2001; Podila et al., 2002; Sundaram et al., 2004) will be expressed at one or more stages of mycorrhizal formation and maintenance, as follows. (i) Genes involved in metabolism (carbohydrate and amino acid metabolism, sucrose metabolism, etc.): similar genes have been shown to be expressed in Eucalyptus-Pisolithus ectomycorrhizas (Voiblet et al., 2001; Duplessis et al., 2005). (ii) Genes involved in vesicular trafficking (vacuolar proteins) and membrane transport, such as ATP-binding cassette, ABC and cation transporters (Menotta et al., 2004). (iii) Genes involved in signal transduction pathways (genes with homology Ras and GTP-binding proteins), as shown by Krüger et al. (2004) for plant genes expressed at preectomycorrhizal stages between Quercus robur and Piloderma croceum. The cross-talk between the symbionts is apparently mediated by these genes (Podila et al., 2002). (iv) Genes involved in protein synthesis and processing (rRNA genes) and gene-expression pathways, which may regulate the metamorphic changes that take place during mycorrhizal development (Podila et al., 2002). As can be seen from Fig. 1(b), 45 cDNA fragments that showed differential expression in any of the combinations tested were either hypothetical proteins or showed no homology to any sequence deposited in the GenBank database. There are two possible explanations for the absence of matching. (i) The sequences could represent 5′ or 3′ untranslated regions (5′ or 3′ UTR). Eliminating this possibility would necessitate finding a full length of each such gene. (ii) Several ectomycorrhiza studies have identified fungal (Voiblet et al., 2001; Podila et al., 2002) or plant (Voiblet et al., 2001; Krüger et al., 2004) genes showing no similarity to any sequences in the NCBI database. Podila et al. (2002) suggested that the expression of these genes, or the genes themselves, may be unique to the particular ectomycorrhizal fungus studied, or that they may represent very rare transcripts that have not been identified and/or characterized previously. We suggest that these sequences could also represent unidentified fungal mycorrhiza-specific genes. A more detailed


study encompassing all of these genes is needed in order to clarify this question. It is clear from our work that the same Cistus plant root clone elicits different responses from different Terfezia isolates (see reaction of fungal isolates 27 and 42a to plant clone M2), and likewise that the same fungal isolate responds differently to different plant root clones (see fungal isolate 27 paired with plant clones M2 and W51), without any apparent correlation to the type of mycorrhiza formed. Our results reveal alterations in the expression of many genes between the two different stages – preinfection and mycorrhiza. Growth under preinfection conditions mainly reflects the effect of soluble cellophane-diffusible root-derived compounds (affected or unaffected by each fungal isolate) on fungal gene expression. On the other hand, progress towards actual formation of the mycorrhiza appears to come to a halt at a certain level, presumably because the required transition to a new gene-expression pattern depends on physical contact and/or on compounds blocked by the cellophane barrier – whether of fungal or root origin. One of the aims of this study was to single out genes responsible for the switch from ecto- to endomycorrhizal type (or vice versa) in our model system. The large group of genes found to be associated with either ecto- or endomycorrhizas occurred under only one or the other of the two growth conditions, suggesting that expression of genes involved in determining mycorrhizal type depends on both conditions and timing (separated growth vs physical contact). These genes could potentially serve as useful markers for either typical ectomycorrhizal or rarer endomycorrhizal structures. Two genes, 221 and 246-1, were associated with ectomycorrhizas under preinfection growth conditions (Table 4), but with endomycorrhizas under mycorrhizal conditions. As such, they appear to be promising subjects in which to study the cellular and molecular events leading to the switch from ectoto endomycorrhizas (or the reverse). As both these genes lacked matches to sequences in the GenBank database, gene extension of the relevant cDNA fragments will have to be performed. We believe these two may prove to be general mycorrhiza-specific markers for both ectomycorrhiza and endomycorrhiza structures. To prove the role of a particular gene in setting an ecto- or endomycorrhizal course, fungal transformation and mycorrhizal experiments would have to be performed with that gene. Moreover, as the plant clone could be the factor that determines mycorrhizal type, plant clone gene expression in the presence of fungal isolates under different growing conditions also needs to be examined. Admittedly, at present we cannot discount the possibility that the differentially displayed fragments isolated in this study do not constitute 63 distinct genes. Some may represent different parts of the same gene. Gene extension of each cDNA fragment will have to be carried out to confirm or eliminate this option.


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Our differentially displayed gene-isolation system tends to under-represent genes expressed during actual mycorrhizal establishment and maintenance (Table 3). It should be kept in mind that the excised cDNA–AFLP fragments were originally isolated as differentially expressed under preinfection growth, compared with genes expressed under free-living conditions, and that although some turned out to be expressed exclusively under mycorrhizal conditions, we may have failed to collect some of the genes present in mycorrhizas. However, the results clearly indicate the transient expression of some genes in preinfection stages, together with alterations in the expression patterns of others. In conclusion, we find several types of fungal genes expressed in mycorrhizas: genes expressed under one of the growth conditions only, either under preinfection (transiently) or mycorrhiza; and genes showing different patterns of expression under different growth conditions. The latter may be involved in choosing the specific cellular pathways that lead to new growth stages. Our results single out, for the first time, some of the genes that may be involved in determining the type of association to be formed: ecto- or endomycorrhizal.

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Supplementary Material The following supplementary material is available for this article online: Fig. S1 Two representative membranes subjected to reverse Northern analysis. This material is available as part of the online article from http://www.blackwell-synergy.com


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