Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088

Research Article Molecular biology

International Journal of Pharma and Bio Sciences ISSN 0975-6299

MOLECULAR MARKER GENES FOR ECTOMYCORRHIZAL

SHIV HIREMATH*1, CAROLYN MCQUATTIE1, GOPI PODILA2 AND JENISE BAUMAN3

1USDA Service, Northern Research Station, Delaware, OH. 2Department of Biological Sciences, University of Alabama, Huntsville, AL. 3Miami University, Oxford, OH.

ABSTRACT

Mycorrhizal symbiosis is a mutually beneficial association very commonly found among most vascular . Formation of happens only between compatible partners and predicting this is often accomplished through a trial and error process. We investigated the possibility of using expression of symbiosis specific genes as markers to predict the formation of ectomycorrhiza. We used antibodies and cDNA probes corresponding to PF6.2 and Lbras genes of bicolor to detect presence and expression of similar genes in other ectomycorrhiza fungi during their association with the red . Analyses using transmission electron microscopy and northern hybridization showed that such genes were expressed in several other fungi forming mycorrhiza with red pine. The results suggest that these genes may have a role in many ectomycorrhizal symbiosis systems and may serve as molecular markers for monitoring symbiosis development.

KEYWORDS: Ectomycorrhizal fungi, symbiosis, Laccaria bicolor, molecular markers.

SHIV HIREMATH USDA Forest Service, Northern Research Station, Delaware, OH.

*Corresponding author

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INTRODUCTION

Most vascular plants, including a variety of identified several factors from the that are , establish mutually beneficial symbiotic involved in the initiation and establishment of relationships with ectomycorrhizal fungi that ectomycorrhizae16,17,18,19. colonize their roots1,2. The interaction between Our previous work has identified several the compatible and fungus pair to the genes from Laccaria bicolor that are turned on in formation of a new complex symbiotic organ response to interaction with red pine called the ectomycorrhiza. The fungi penetrate roots16,17,20,21,22. Using an in vitro interaction the and grow intercellularly system we observed drastic changes in the gene between cortical cells forming a network of expression of the fungal partner in which a hyphae. The structure includes the innermost number of genes were activated. Among several Hartig-net in the root cortex, the fungal sheath or genes characterized using this method, the mantle on the surface of the root, and the PF6.2 and Lbras genes have been appeared to hyphae extending into the soil3. Extensive be very important since the degree of change in nutrient exchange takes place between the plant expression level was significant. Moreover, and fungal partners through this ectomycorrhizal expression of these genes was required for organ2,3. The fungus acquires from the initiation, establishment as well as maintenance plant in the form of hexoses4,5,6 while the plant of the functioning mycorrhiza16,17. The Lbras was obtains minerals through the fungus by closely related to ras family of genes, which have mobilizing substrates7,8,9. By extending the been shown in other systems to be involved in absorptive capabilities of the root system, the signaling pathways controlling and mycorrhizal association has been shown to proliferation. The function of the PF6.2 gene is improve plant’s ability to tolerate biological and unknown;its; sequences did not match to environmental stresses, including challenge by anything reported and deposited in the Genbank, pathogenic fungi, heavy metal , although it showed some similarity to signal excessive erosion, , and reduced pH of transduction genes. However, PF6.2 was one of the . The ectomycorrhizal phenomenon, the most abundantly expressed genes in essential for health and survival of many conifers response to exposure to plant . Since and hardwood , has been studied widely mycorrhizal symbiosis is a unique process from ecological and physiological perspectives. involving formation of a new organ common to A myriad of biochemical events happen leading plant and fungus, it is possible that PF6.2 to the initiation, formation and establishment of a represents a unique gene not found in other functioning mycorrhiza. These events include systems reported. The complete genome turning on and off of several genes in the fungus sequence of the L. bicolor reported recently as well as in the plant10,11. Presumably, the confirms that symbiotic fungi such as L. bicolor root cells produce certain elicitors that induce contain several unique genes not found in other expression of fungal genes involved in organisms18. Temporal expression studies ectomycorrhiza establishment10,12,13. These showed that both PF6.2 and Lbras were induced include genes responsible for triggering very early during interaction with the plant roots attachment and developmental processes and continued to be expressed in established leading to the formation of Hartig net and hyphal . Transmission electron microscope mantle14,15. In addition, certain fungal genes (TEM) analyses using Lbras and PF6.2 involved in eliciting host plant defense reaction antibodies showed that these were may be turned off to initiate the association present in both mantle and Hartig-net tissues process. Although all the molecular mechanisms and were localized near the cell wall16,17. involved have not been understood, studies Although many plants associate with undertaken in last couple of decades have ectomycorrhizal fungi, it is not possible to predict

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 which plant species will form ectomycorrhiza with prepared by placing mycelial pieces grown on a given ectomycorrhizal fungus. There appears M4N agar plates for 2-3 weeks at 240 C in a 1 L to be a “compatibility” factor for successful M4N media contained in a 2 L Erlenmeyer flask formation of the symbiotic unit. However, there and incubating at room temp in the dark for 2-3 are no known quick methods to determine this weeks. The media was then macerated using a compatibility and, often, this is accomplished by Tissumizer homogenizer for 30 sec and used as a trial and error method. In order to investigate an inoculum. About 3 months after inoculation, whether genes like PF6.2 and Lbras are specific roots were examined under microscope for the only to L. bicolor X red pine system or present in presence of mycorrhizae. The mycorrhizal all mycorrhizal symbiosis systems, we undertook tissues were collected and used for TEM the present study. Our preliminary studies using analyses as well as for isolation of RNA for antibodies to L. bicolor PF6.2 and Lbras proteins northern analyses. A portion of the tissue was had suggested that similar proteins may be used for isolating DNA and to identify the fungus present in other mycorrhizal fungi associating present in the ectomycorrhizae. with red pine. Therefore, we looked at expression of these genes in five other DNA isolation, PCR and mycorrhizal associations with red pine. Our Identification of the fungus results indicated that such genes were indeed The ectomycorrhizal tissues were used to isolate activated in other systems also, suggesting that fungal DNA and identify the fungus through ITS- PF6.2 and Lbras have a role in all region sequence as described earlier25,26. Briefly, ectomycorrhizal symbiosis systems and may about 10 mg tissues were homogenized in serve as molecular markers for monitoring extraction buffer using a bead beater for 3 min symbiosis development. and DNA extracted using QIAgen’sDNAeasy Plant Mini Kit. The DNA was used in a PCR MATERIALS & METHODS using primers that amplify the highly variable MEDIA AND CULTURES internal transcribed spacer (ITS) region of the fungal ribosomal DNA. The PCR product was

either used for nucleotide sequencing directly or The fungal cultures of rubescens, was cloned first and then sequenced. By Laccaria laccata, Suillusluteus, comparing the sequence obtained with those Suillusamericanus, Rhizipogon present in the Genbank the identity of the fungus rubescensandLaccaria bicolor were isolates was ascertained27. associated with the roots of red pine obtained

from Michigan’s Upper Peninsula (provided by D. Protein and Antibodies Richter, School of , Michigan Antibody production to Lbras protein has been Technological University, Houghton). The fungi described earlier17. The antibodies to PF6.2 were grown and maintained on MMN medium as protein were produced using a similar method. previously described20,23. Briefly, the cDNA fragment corresponding to

PF6.2 was cloned into the expression vector Formation of mycorrhizae pet22b+ (Novagen), which facilitated expression This was done essentially as described 21,24 of a fusion protein containing a 3’-histidine earlier . Briefly, red pine (Pinus resinosa) tag28,29,30. After transformation of E. coli with this seedlings were grown in a sterile container (450 construct and expression of proteins according ml) by planting seeds in a 3:1 vermiculite: to the manufacturer’s instructions, the PF6.2 sphagnum peat containing 0.1X M4N medium fusion protein was purified from cell extracts without glucose. After the seedling had grown for through affinity chromatography using the His- ~ 3months and developed transverse roots, Bind Metal Chelation resin (Novagen). Polyclonal fungal culture was introduced by adding a 30 ml antibodies to the fusion proteins were generated aliquot of liquid inoculum. The latter was

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 in rabbits as described earlier17,31. The analysis was performed essentially as described antibodies were tested for their specificity earlier16,17. Total RNA (10 Ng) was through western blot analysis17. electrophoresedon 1% agarose gels and transferred to Hybond-N membranes(Amersham Immunolocalization with Transmission Pharmacia Biotech, Piscataway, NJ).Gels were Electron Microscopy (TEM) stained withSYBR Green to determine equal Sample preparations for TEM analyses32 were loadings and intensity ofRNA.The cDNA similar to what was described earlier17. Briefly, fragments of Lbras and PF6.2 cDNA clones were small (< 4 mm) mycorrhizal and nonmycorrhizal isolated and purified by agarose gel root pieces were cut and fixed in 2.5% electrophoresis followed by extraction of DNA glutaraldehyde present in buffer NaP (10 mM using the QIAEX gel extractionkit (Qiagen Inc., sodium , pH 7.2) by incubating for 16- CA). About 25 ng of purified cDNAwas labeled 20 h at 40°C. They were then placed under with 32P-dCTP (3000 Ci/mmol, Amersham Corp, vacuum at room temperature for 2 h. After NJ) using the DECAprime II DNA Labeling Kit washing (3 × 15 min) with NaP buffer, samples (Ambion, Austin, TX,U.S.A.). These were used were treated with 1% osmium tetraoxide for 1 h as probes in hybridization analyses ofthe and washed with water. Water content of the membrane-bound nucleic acids33,34,35. samples was gradually removed by successive Prehybridization, hybridizationand washings washes with 30% ethanol, increasing the ethanol were done as described before16. concentration gradually. After a final treatment with 100% ethanol, infiltration withLR white resin RESULTS 32 was performed . An ultramicrotome equipped with a diamond knife was used to get cross The PF6.2 in L. bicoloris sections of roots (100 to 120 nm) on grids initiated very early during interaction of the that were subjected to immunological analysis. fungus with the red pine roots. The PF6.2 mRNA After an initial blocking step in TBS (50 mMTris- encoding a protein of about 47 kDwas ~ 1.5 kb in HCl, pH 7.6, containing 0.5 M sodium chloride) length and could be detected as early as 6 h into containing 1% bovine serum albumin (BSA) and interaction16. The Lbras mRNA from L. bicolor on 1:30 dilution of rabbit preimmune serum, the the other hand was detectable only 48 h after grids were incubated for 16 to 20 h with 1:1000 interaction with roots. The Lbras mRNA was also dilution of anti-Lbras antiserum or anti- ~ 1.5 kb in length, but encoded a protein of PF6.2antiserum in TBS at room temperature. about 26 kD17. However, both PF6.2 and Lbras After washing with TBS, they were treated with continued to be expressed in established TBS containing 1% BSA and 1:20 dilution of mycorrhizas in L.bicolor X red pine. Therefore, gold-labeled second antibody (goat antirabbit we used established mycorrhizal tissue for immunoglobulin Polygold 10 nM; Polysciences, analyses in these experiments. Warrington, PA, U.S.A.). After incubation for 1 h, root sections were washed several times with 1. Ectomycorrhiza formation TBS followed by water and dried. Some of the The ectomycorrhiza synthesis performed with samples were subjected to post-staining with red pine seedlings under semi-sterile conditions 32 uranyl acetate . The grids were then examined is shown in Figure 1. Seedlings were typically with a JEOL JEM-1010 TEM operated at 80 kV. about 7-10 cm in height and had developed lateral roots 3 months after . After Northern analyses inoculation and formation of ectomycorrhiza Mycorrhizal root tissues were ground to a fine (about 3 months later), their height was about powder in liquid and total RNA was 12-15 cm. The ectomycorrhizal association was isolated using the Trizolkit (Gibco BRL, MD) as not widespread on the root per manufacturer’s instructions16. Northern

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Figure1 Synthesis of ectomycorrhiza: Red pine (Pinus resinosa) seeds were germinated in vermiculite medium in a sealed container under semi-sterile conditions. After ~3 months, when the seedling developed lateral roots, the ectomycorrhizal inoculum was introduced and incubated for ~ 3 months for ectomycorrhiza to be formed. system under the conditions we used (only about 15-25%). We checked the roots under the microscope to select portions of tissue for further analysis. In order to confirm that the fungus in the ectomycorrhizal tissue was the one that was used for inoculation, we reisolated the fungus from the tissue and were subjected to morphological analysis.

2. Antibodies and transmission electron microscopy Based on the nature, expression pattern, and the level of expression of PF6.2 and Lbras in L. bicolor, we had predicted that similar genes may be present in other ectomycorrhizal systems. Our preliminary studies using the antibodies to L. bicolor PF6.2 showedthat such proteins were indeed present in other ectomycorrhizal fungi. We confirmed this by performing immuno-dot blot analysis using ectomycorrhizal tissues of red pine with the fungi Amanita rubescens,

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Figure 2 Analyses with antibody to the Lb PF6.2 protein. TEM photographs of mantle and Hartig-net hyphae from fungal X red pine ectomycorrhizas. The fungus and the tissue type are indicated on each picture. Immunoreactive material indicated by gold label (seen as black dots) was localized primarily near fungal cell walls (arrows). H- Hartig-net ; M- Mantle hypha; W- Root ; RC- electron-dense root material.

Laccaria laccata, Suillusluteus, tissues are shown in Figure 2. The gold label, Suillusamericanus and Rhizipogon rubescens which indicates the presence of the (data not shown). All these fungal extracts immunoreactive protein, is seen as an opaque showed presence of proteins that cross reacted dot in the TEM analysis (shown by arrows). The with antibodies to Lbras and PF6.2. Therefore, analyses indicated that in all the tissues we used these antibodies in TEM analyses to examined there was a protein cross-reacting localize their presence in ectomycorrhizal with the PF6.2 antibodies. Although the figure tissues. The TEM results obtained from shows data obtained with only some of the immunolocalization of PF6.2 in mycorrhizal tissues, we observed proteins cross reacting

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 with the antibodies in all ectomycorrhizal tissues nonmycorrhizal tissues (only root) treated and from Amanita rubescens, Laccaria laccata, observed in a similar fashion, no Suillusluteus, Suillusamericanus and immunoreactive material was detected (data not Rhizipogon rubescens. This included: 1) the shown). A similar pattern was seen in TEM Hartig-net (H), the network of hyphae analyses using the Lbras antiserum (Figure 3). penetrating in the interstitial space of the The figure shows only some of the data cortical cells of the roots, and 2) the mantle (M) obtained with antibodies to Lbras protein. where the fungal hyphae are on the surface of However, both mantle and Hartig-net tissues of the roots and extending from there. In all cases, all the fungi tested had the immunoreactive the immunoreactive material was primarily material. Also, as in the case of PF6.2, the label localized near the cell wall as observed earlier was mostly present near the cell wall (indicated in the case of L. bicolor17. However, in case of by arrows).

Figure 3 Analyses with Antibody to the Lbras protein. TEM photographs of mantle and Hartig net hyphae from fungal X red pine ectomycorrhizas. The fungus and the tissue type are indicated on each picture. Immunoreactive material indicated by gold label (seen as black dots) was localized primarily near fungal cell walls (arrows). H- Hartig net hypha; M- Mantle hypha; W- Root cell wall; RC- electron-dense root material.

We also looked at the presence of respective blot hybridizations had shown that L. bicolor mRNAs in the ectomycorrhizal tissues. Similar cDNAs probes were able to hybridize to RNA in to antibodies, our preliminary studies using dot other fungi, presumablytheir counterparts (data

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 not shown). Our earlier work had also shown panel shows the SYBR Green II stained gel thatwhile PF6.2 mRNA was detectable in L. picture before nucleic acids were transferred to bicoloras early as 6 h after interaction, the Lbras the Hybond membrane and the top panel shows mRNA was observed only 48 h after interaction. the autoradiogram of the hybridized membrane. However, both continued their expression in the As expected, we were able to detect the ~ 1.5 established mycorrhiza. Therefore, we decided kb PF6.2 mRNA from L. bicolor as reported to use the completely formed mycorrhizal earlier16. The probe also hybridized to a 1.5 Kb tissues for these analyses. Also, we used total RNA present in each of the fungal RNA RNA from each tissue for analysis since analyzed. The size of the band in each case separating poly(A) RNAwould have required was similar to the size of the PF6.2 mRNA from larger quantities of tissue, which was not L. bicolor. While the relative intensities of available in this study. Also, the sensitivity of the hybridization signal were same among most technique was adequate to detect the RNA samples, only in case of R. rubescensit corresponding sequences in the total RNA16,17. was lower. This was not due to the total amount However, considering low degree of homology of RNA used in the analysis as the stained gel that may exist among cDNA/mRNA sequences, indicated that all the lanes had similar amounts we sued less stringent conditions for of RNA. The observed result was probably hybridization analyses. Figure 4 shows the because of R. rubescens PF6.2 having a lower northern analysis results obtained using the degree of homology to the L. bicolor PF6.2. PF6.2 cDNA fragment as the probe. The lower

Figure 4 Northern analysis using PF6.2 cDNA probe.TotalRNAwas(107g) waselectrophoresed on 1% agarose gels and RNA bands were visualized by staining with SYBR (Lower panel). The bands were transferred to a Hybond membrane and subjected to hybridization using labeled PF6.2 cDNA probe as described in Materials and Methods. RNA size markers used in the analysis can be seen in the left lane.

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We also looked at the ras mRNA levels in these these tissues had an equivalent of Lbras mRNA tissues using the Lbras cDNA fragment as which was being expressed in the functional probe in northern analysis (Fig. 5). Results were ectomycorrhiza. Moreover, as seen with PF6.2 very similar to those observed for the PF6.2 mRNA, the Lbras mRNA also appeared to have northern analysis. In case of L. bicolor, the less homology to its counterpart in R. probe hybridized to the 1.5 kb Lbras mRNA as rubescens. The signal was much lower than reported earlier (Sunderam et al 2001). The others, although same amounts of total RNA probe also hybridized to a ~1.5 kb RNA present were used for the analyses. in each mycorrhizal tissue, suggesting that

Figure 5 Northern analysis using Lbras cDNA as probe. Total RNAs from each of the mycorrhizal tissue was subjected to northern analysis as described in Materials and Methods. For details see legend to Figure 4.

DISCUSSION

The mycorrhizal symbiosis represents an and forestry. Initial analysis of the unique system where two different organisms genome revealed several unexpected features interact to form an entirely new organ in the L. bicolor genome which contained a mycorrhiza, which then becomes common to battery of unique effector-type small secreted both organisms. Both root cells and fungal cells proteins with unknown function18. Further are integrated in this organ and function analysis showed that at least one such protein together to transport nutrients to each other. It is MISSP7, expressed in high copy numbers conceivable that such a complex symbiotic unit during interaction with the plant roots, may be would involve genes and mechanisms that are essential for symbiosis development36. It very distinctive. Recently, entire genome appears similar genes are required for initiation, sequences of two symbiotic fungi Laccaria development and maintenance of the bicolor and were mutualistic symbiosis37. Our interest was to see reported; this study was undertaken because of whether we could identify and usesimilar the importance of mycorrhizal symbiosis in symbiosis specific genes that may be common

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 among all mycorrhizal systems and their fungi and are required for mycorrhizal possible use as markers for monitoring symbiosis. symbiosis development. We chose PF6.2 and We used a sterile containerized system Lbras genes for this study based on our for generating the mycorrhizal tissues. We have observations of their expression during initiation consistently used this system to produce and establishment of mycorrhiza symbiosis in mycorrhizal tissues without problems of any case of L. bicolor X red pine16,17,21. Similar to contamination. In the present study, the the MISSP7, the PF6.2 and Lbras were ectomycorrhizal tissues we used for analysis expressed at high levels during interaction with indeed contained the same fungus that was plants. PF6.2 was one of the most abundantly used for inoculation. We conformed this by expressed genes among all the symbiosis- isolatingthe fungus back from the mycorrhizal specific genes we looked at during interaction of tissues and performing morphological analyses. L. bicolor with red pine roots. It was expressed In other instances, we have used the same as early as 6 hours after exposure to roots in system where identification of the fungus was the in vitro interaction studies20,21. Moreover, the done by isolating the fungal DNA followed by expression continued even during analysis of the variable ITS region of the fungal established/functioning mycorrhiza indicating ribosomal region25,26,27. Routinely, we found the that it serves an important function in same fungal species that was used in mycorrhizal symbiosis. It was a unique gene inoculation in such experiments. Our data not related to any other genes found in other clearly indicates that indeed the fungi tested systems.The complete nucleotide sequence of here had similar proteins present which were the PF6.2 gene revealed an unusual structure, closely related to their counterparts in L. bicolor. which had multiple repeats within their exons as Since we were not able to isolate enough well as introns. Presence of such repeats protein for generating antibodies, we had to suggested that the PF6.2 may have a function resort to in vitro expression followed by similar to some of the genes associated with purification of respective proteins in . signal transduction and recognition domains of The antibodies generated were specific to the receptors as well as metal binding and transport protein, which was confirmed by western proteins38,39,40. Based on all observations it was analysis. Antibodies did not cross react with any clear that not only its expression was symbiosis protein in root tissues alone and in L. bicolorthat specific, it probably played an important and had not been exposed to roots, indicating that critical role in the establishment and they were specific to respective maintenance of the symbiosis.Similarly, the proteins.Therefore, we employed these in TEM Lbras belongs to ras family of genes, which are analyses. The gold spots indicative of the known to be associated with signaling pathways presence of respective proteins were present in controlling cell growth and proliferation. Recent both Hartig-net and mantle tissues in all the work on L bicolor genome has shown that it mycorrhizal tissues. Furthermore, as in the case belongs to a group of several RAS GTPases, of L. bicolor, these proteins were localized near which are key components of signal the cell wall in the mycorrhiza17. In mammalian transduction pathways37. It was not surprising cells, ras proteins are localized near the plasma that sucha gene is involved in the development membrane and interact with tyrosine kinase of mycorrhiza. The Lbras from L. bicolor was receptors41 to influence the cellular signaling closely related to mammalian ras genes and pathways controlling critical cellular events. was able to complement the ras 2 in S. Since PF6.2 also appears to be related to signal cerevisiae17. Considering the characteristics of transduction pathway genes, it makes sense PF6.2 and Lbras, we thought similar genes that it too has to be localized near the cell wall would most likely be present in all mycorrhizal and closer to the inter-cellular region in the ectomycorrhizal tissue. Taken as a whole, it

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Int J Pharm Bio Sci 2013 Jan; 4(1): (B) 1075 - 1088 appears that as in L. bicolor, the counterparts of optimum utilization of the mycorrhizal the PF6.2 and Lbras in other fungi have similar phenomenon. Biochemical markers linking soil function. Presence of homologous genes in chemistry and individual/group of fungi that will other fungi was further confirmed by the be compatible under certain conditions will help northern analyses. It is not clear what degree of in determining suitable fungi that will be homology exists among the L. bicolor genes successful in a specific region. At present, there tested here and their counterparts detected are no biochemical methods to determine this from other fungi. In the TEM analyses, we could compatibility. The studies described here not see any quantitative differences. However, suggest that PF6.2 and Lbras like genes have a the northern analysis suggested that the role in all ectomycorrhizal symbiosis systems homology was adequate to detect respective and are important for functioning of mRNAs using the technique. Though we used ectomycorrhiza. Such genes can be used for moderately stringent conditions for hybridization monitoring formation, development and analyses, results showing a single specific band functioning of ectomycorrhizas and will be in each fungal RNA samples suggest that there useful in identifying and better predicting the is significant homology among L. bicolor cDNA tree/fungus combinations that would work under and its counterparts in other fungi tested. specific conditions. However, this was partially true in the case of rubescens. Both PF6.2 as well as CONCLUSION Lbras cDNA probes hybridized weakly to their respective counterparts from this fungus (Figure The fungi Amanita rubescens, Laccaria laccata, 4 and Figure 5). However, overall similarities Suillusluteus, Rhizopogon rubescens and such as presence and localization of Suillusluteus appear to have similar proteins homologous proteins cross reacting with (PF6.2 protein and Lbras protein), which cross- antibodies in TEM analyses as well as reacted with the antibodies raised against their sequence similarities seen by northern analyses counterparts from Laccaria bicolor. The label suggest that all the fungi tested have genes was localized mainly near the fungal cell wall as similar to PF6.2 and Lbras in their genome. observed in L. bicolor X red pine Isolation of respective genes from these fungi ectomycorrhizas. This was true for both the and sequence characterization will be PF6.2 and the Lbras proteins necessary to confirm this. There is a lack of antibodies.Northern hybridization analyses basic biochemical knowledge describing what using cDNA probes corresponding to PF6.2 and factors contribute to successful formation of Lbras showed that other fungi had similar symbiosis and, therefore, the success of mRNAs that were expressed in reforestation through mycorrhizal phenomenon. ectomycorrhizae with red pine roots.The results Very often, the process of utilization of suggest that these genes may have a role in mycorrhizal fungi is a trial and error process. many ectomycorrhizal symbiosis systems and Adequate knowledge of relationships between may serve as molecular markers for monitoring soil conditions and tree/fungus interactions has symbiosis development. not been worked out, which is very important for

ACKNOWLEDGMENT

The authors thank Kirsten Lehtoma for technical support and assistance for some of this work.

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