J Plant Physiol. Vol. 141. pp. 2-6 {1992}

Introduction

Comparison of Acid Soluble Protein Synthesis in Roots of Endomycorrhizal Wild Type Pisum sativum and Corresponding Isogenic Mutants

LUISA SCHELLENBAUM*, SILVIO GIANINAZZI\ and VIVIENNE GIANINAZZI-PEARSON

Laboratoire de Phytoparasitologie, INRA-CNRS, Station de Genetique et d' Amelioration des Plantes, INRA, BV 1540, 21304 Dijon cedex, France

* Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA 1 To whom correspondance should be addressed.

Received April 3, 1992 . Accepted July 20, 1992

Summary

The aim of this study was to compare profiles of acid soluble proteins in roots of Pisum sativum L. cv. Frisson and non-mycorrhizal mutants (myc-) when infected by vesicular arbuscular mycorrhiza funghi. Quantitative and qualitative modifications were detected by native or SDS PAGE in the protein composi­tion of acidic extracts of endomycorrhizal roots of myc+,nod+ wild type P. sativum and of a myc+,nod­mutant, as compared to uninoculated control plants or a myc-,nod - mutant, whether this was inoculated or not with Glomus mosseae. In native PAGE two proteins, already present in extracts of non-mycor­rhizal pea roots, were enhanced in G. mosseae-infected myc+ plants (nod+ or nod-). One protein which appeared after inoculation with G. mosseae or G. /asciculatum in the roots of all pea plants, including the myc-, nod- mutants, may be associated with appressorium formation. Four new proteins were detected in root extracts of G. mosseae-infected myc+ peas (nod+ or nod-), as compared to the myc-, nod- mutant or uninoculated controls. One of these was probably of fungal origin, since it was also found in G. mos­seae-infected tobacco and leek roots. These plants showed protein modifications different to those in endomycorrhizal peas, suggesting host-specific responses. SDS PAGE revealed the presence of two poly­peptides of about 25 and 35 kDa in endomycorrhizal roots of myc+ peas. None of the endomycorrhiza­related proteins detected in roots corresponded to those extracted from spores of different fungi. This study provides new evidence that different molecular modifications occur during successful (myc+ pea, leek, tobacco) and unsuccessful (myc- pea) endomycorrhiza infections, some of which are host-de­termined and others related to the expression of the fungal genome.

Key words: Pisum sativum, Glomus species, Acaulospora laevis, endomycorrhiza, myc-nod- mutants, soluble proteins.

Abbreviations: PAGE = Poly Acrylamide Gel Electrophoresis; PVPP = PolyVinylPolyPyrrolydone; SDS = Sodium Dodecyl Sulphate.

The molecular basis of fungus-plant interactions in mycor­rhizal associations with regards to changes in symbiont geno­me expression during establishment of the symbioses is poorly understood (Gianinazzi, 1991; Bonfante-Fasolo and

Perotto, 1992). Higher transcriptional activity is indicated by an enhanced activity of the host nucleus following my­corrhizal infection (Berta et aI., 1990), and new mycorrhiza­specific polypeptides or proteins have recently been shown to be produced in ectomycorrhiza (Hilbert et aI., 1988, 1991) or in vesicular arbuscular endomycorrhiza (Dumas et al.,

© 1992 by Gustav Fischer Verlag, Stuttgart

1989, 1990; Pacovsky, 1989). Some endomycorrhiza-specific polypeptides have also been reported to immunologically cross-react with antibodies against nodule-specific proteins (nodulins) (Wyss et ai., 1990). However, nothing is known about the function of these mycorrhiza-specific polypeptides and proteins, or whether they playa role in interactions be­tween the plant and fungal symbionts.

Recently, chemically induced isogenic mutants of Pisum sa· tivum have been discovered (Duc et ai., 1989, Gianinazzi­Pearson et ai., 1991) that are either resistant to both endomy­corrhizal fungi and Rhizobium (myc- ,nod-), the fungal sym­biont only reaching the morphological stage of appressorium development, or establish an endomycorrhizal association but do not nodulate (myc+,nod-).

The aim of the present study was to compare the soluble protein profiles of roots of endomycorrhizal and non-my­corrhizal wild type (myc+ ,nod +) P. sativum with those of the different mutants (mye,nod- and myc+,nod-), in order to

detect possible molecular changes specific to the symbiotic state. Root soluble proteins have also been analyzed in P. sa· tivum infected with different endomycorrhizal fungi, and in different plant genera infected with the same fungus as P. sa· tivum. Since vesicular arbuscular fungi cannot be grown in pure culture, spore soluble proteins were extracted to see whether any new endomycorrhiza-related proteins were al­ready present in spores and therefore likely to be of fungal OrIgm.

Materials and Methods

Plant material and growth conditions

Seeds of Pisum sativum L. cv. Frisson (myc+ ,nod+) and of the corresponding P56 (myc+,nod-) and P2 (myc- ,nod-) mutants were surface sterilized in 7 % calcium hypochlorite for 10 min, preger­minated in vermiculite in the dark at 20 0 e and planted into pots containing 400g of a disinfected clayloam soil (pH 7.6): perlite (3: 1) mix. Plants were either inoculated with Glomus mosseae (Nicol & Gerd) Gerd and Trappe (isolate LPA 5) using a root inoculum from a stock culture of Allium porrum L. cv. Verina, or grown in disin· fected soil as non-mycorrhizal control plants. All plants were inocu· lated with Rhizobium leguminosarum bv. viciae, strain 1007 (from N. Amarger, INRA, Dijon). Additional plants of wild type P. sati· vum (myc+,nod+) were inoculated with G.fasciculatum (LPA 7) and Acaulospora laevis Gerd and Trappe (LPA 1), using a silty acid soil pH 5.0) for the latter. Nicotiana tabacum L. cv. Xanthi and A. por· rum cv. Verina seeds were sterilized and inoculated with G. mosseae as described above. Plants were maintained in a growth chamber (20 0 e, 80% rh, 16h day, 300llmols- 1 m- 2

) and watered daily with deionized water.

Extraction of soluble proteins from roots and spores

Roots from control and inoculated plants were harvested three weeks after transplanting (except for tobacco and leek which were harvested at 6 weeks), carefully washed in cold water and root pieces with visible nodules were removed. Samples were ground at 4 °e in 0.06 M phosphate-citrate buffer (1 g mL -1) at pH 2.8, containing 5 mM ascorbic acid, 1 % PVPP and 15 mM mercaptoethanol. The samples were centrifuged (60 min 10000 x g) and the supernatant collected. Spores of G. mosseae (400), G. fasciculatum (400) and A. laevis (200) were isolated from soil by wet sieving. Soluble spore

Protein in endomycorrhizal myc+ and myc- plants 3

proteins were extracted in 100 ilL of buffer at pH 7.6 according to Sanders et al. (1992), and at pH 2.8 using the same buffer as that used for roots. The protein contents of the extracts were estimated by the method of Bradford (1976), using bovine serum albumin as a stand­ard.

Electrophoretic separation of proteins

Soluble proteins were separated at 4 °e using discontinuous na­tive (Davis, 1964) and SDS (Laemmli, 1970) PAGE on mini vertical uniform polyacrylamide slab gels (Imm thick, 8x7cm) at 10% (25 rnA h -1) and 15 % (30 rnA h -1), respectively. Polypeptide mobili­ties were compared with those of a range of molecular weight mark­ers (Sigma kit). The same volume of extract was used for all samples (10 ilL for roots, 15 ilL for spores). Proteins were revealed by silver nitrate staining (Blum et aI., 1987).

Determination of the endomycorrhizal infection

The amount of infection developing in roots was estimated ac­cording to Trouvelot et al. (1986), after digestion of roots in 10% KOH and staining with cotton blue (0.1 %) in lactic acid. Parame­ters are expressed as frequency of infected root samples (F%) and percent colonised root cortical length (M%) within the endomycor­rhizal root system.

Results and Discussion

Endomycorrhizal infection and arbuscule formation was already well-established in the three week-old wild type (myc+,nod+) and PS6 mutant (myc+,nod-) pea plants, and values for F% (77 -100) and M% (29 - 55) did not differ sig­nificantly for G. mosseae or G. /asciculatum infections in re­peated experiments. Significantly lower values (P = 0.05) were, however, obtained for infection of P. sativum (myc+,nod+) inoculated with A. laevis (F% = 42, M% = 4). In the case of G. mosseae-inoculated mye ,nod- mutant, the frequency of root fragments with appressoria was consist­ently high (average of 78 %).

Endomycorrhizal root extracts of P. sativum cv. Frisson contained more soluble proteins than non-mycorrhizal ones, as reported in preliminary analyses (Schellenbaum, 1991) and by Dumas et al. (1989) for endomycorrhizal tobacco roots. In fact, in the present study more protein was detected fol­lowing PAGE of pH 2.8 extracts of endomycorrhizal roots of wild type P. sativum (myc+, nod+) and of the PS6 mutant (myc+,nod-), as compared to uninoculated controls and to

the P2 mutant (myc-,nod-), whether this was inoculated or not with G. mosseae (Fig. 1 a and b). Protein bands were par­ticularly intense in the upper part of gels of en do mycorrhizal root extracts of myc+ pea plants (wild type and PS6). Both quantitative and qualitative modifications were observed in protein bands for the different extracts and a certain number of these could be clearly distinguished (Figs. 1 a and 2 a).

Two proteins already present in extracts of non-mycor­rhizal pea roots, and indicated by arrows at Rfs 0.86 and 0.88 in the lower part of gels, were enhanced in G. mosseae-in­fected myc+ plants, whether these were nod + or nod - (Fig. 1 a). One band at Rf 0.40 (E2) was present in the roots of all endo­mycorrhiza-inoculated pea plants, including the mye,nod­mutant, but it was absent from the uninoculated controls

4 LUISA SCHELLENBAUM, SILVIO GIANINAZZI, and VIVIENNE GIANINAZZI-PEARSON

a

b

94 67

kD

43 -30 -20.1-

14.4-

Myc+

Nod+

NM Gm

Myc+

Nod+

NM Gm

Myc+

Nod­

NM Gm

Myc+

Nod­

NM Gm

(Fig. 1 a and 2 a). It was therefore linked to the presence of the different fungi and since these do not penetrate the root tissues of myc- plants, this protein could be associated with the formation of appressoria during the initial stages of the endomycorrhizal infection. Four new protein bands were present in extracts of roots from myc+ plants infected by G. mosseae, when compared to extracts of uninoculated controls and of the myc-,nod- (P2) mutants. These proteins are in­dicated as E1 (RfO.34), E3 (RfO.61), E4(0.91) andES (RfO.97) in Fig. 1 a and as they were in both the myc+ ,nod- and myc+,nod+ root extracts, they are probably specific to an ac­tive, well-established endomycorrhizal symbiosis. Another protein band (R) at Rf 0.54 (Figs. 1 a and 2 a) was only de­tectable in root extracts of myc+,nod+ plants (mycorrhizal or non-mycorrhizal) and was absent from myc+,nod- plants suggesting that it is directly linked to the presence of Rhizo­bium. Since all root pieces with visible nodules had been re­moved from the myc+,nod+ plants, this protein could be re-

Myc­

NOd­

NM Gm

Myc­

Nod­

NM Gm

Fig. 1: Soluble protein patterns of 3 week­old roots of Pisum sativum cv. Frisson (myc+,nod+), P56 (myc+,nod-) and P2 mu­tants (myc- ,nod -), either uninfected (NM) or inoculated with Glomus mosseae (Gm) and separated by (a) native 10% PAGE, (b) SDS 15 % PAGE.

lated to early stages of the Rhizobium infection, as described for nodulins (Govers et al., 1987).

Quantitative and qualitative changes in protein composi­tion were confirmed between root extracts of endomycor­rhizal (myc+,nod+; myc+,nod-), control and myc,nod- mu­tant plants under denaturing conditions of SDS PAGE (Fig. 1 b). Two new polypeptides (indicated by arrows) were detected in infected roots of the myc+ (nod+ or nod-) plants and were absent from myc- (P2) root extracts and uninocu­lated controls. Comparisons with molecular weight markers indicated that these two polypeptides were of about 25 and 35 kDa, respectively. Their presence only in the infected myc+,nod+ and myC+,nod- plants suggests that their syn­thesis is specific to the endomycorrhizal symbiosis.

Infection of myc+ ,nod+ pea plants with G. /asciculatum or A. laevis gave slightly different soluble protein profiles in na­tive gels to that observed in G. mosseae-infected roots (Fig. 2 a). Only the new proteins E1, E2, E3, R and the enhanced pro-

a

b

Gft

P. sativum

Gm AI

A. porrum

NMGm

_ El

- E2

- R

Rf

0.48 0.52 0.56

E4

E3

N. tabacum

NMGm

Fig. 2: Native soluble proteins from (a) 3 week-old Pisum sativum cv. Frisson roots inoculated with Glomus Jasciculatum (Gft), G. mos­seae (Gm) or Acaulospora laevis (AI) and from (b) 6 week-old Nico­tiana tabacum and Allium pm'rum roots, either uninfected or inocu­lated with G. mosseae.

tein at Rf 0.88 were detectable in the presence of G. Jascicu­latum, whilst protein modifications were even less striking in the A. laevis infections as compared to the other host-fungus combinations, possibly due to the low level of infection de­veloped by A. laevis in P. sativum cv. Frisson. A new band at the same Rf as E4 was found in root extracts of six week-old G. mosseae-infected tobacco (F% = 95, M% = 20) and leek (F% = 79, M% = 49). Since this protein band was also pre­sent in myc+ pea roots infected by the same fungus, it seems highly probable that this protein is of fungal origin.

As in endomycorrhizal pea roots, soluble protein synthesis was enhanced both quantitatively and qualitatively in endo­mycorrhizal leek and tobacco roots (Fig. 2 b). At least three new protein bands appeared in leek and two in tobacco roots infected by G. mosseae, in addition to the band correspond-

Protein in endomycorrhizal myc+ and myc" plants 5

a

b

pH 2.8

AI Gft Gm

pH 7.6

AI Gft Gm

Fig. 3: Native soluble proteins extracted from Acaulospora laevis (AI), Glomus Jasciculatum (Gft) or G. mosseae (Gm) spores at pH 2.8 (a) and 7.6 (b).

ing to E4. Two bands showing the same mobilities in ex­tracts of both plants (Rfs 0.48 and 0.56) were not present in mycorrhizal pea roots, whilst one additional band (Rf 0.52) was specific to infected leek roots and it is therefore probable that some of these modifications represent host-specific re­sponses.

Only a few proteins were detected after electrophoretic separation of A. laevis spore proteins soluble at pH 2.8 and none could be shown for C. Jasciculatum or G. mosseae (Fig. 3 a), even when a large number of spores were extracted (900 spores 100 IlL"1, results not shown). Although more proteins, including some of C. mosseae, could be detected by extracting at pH 7.6 (Fig. 3 b), none corresponded to those appearing in root extracts after endomycorrhizal infection. These observations suggest that the newly detected proteins reported in the present work in endomycorrhizal myc+ pea, leek or tobacco roots are not present in spores. Proteins which are thought to be of fungal origin may be specifically

6 LUISA SCHELLENBAUM, SILVIO GIANINAZZI, and VIVIENNE GIANINAZZI-PEARSON

induced, or enhanced, during the symbiotic phase of fungal development within the host tissues.

The results presented here show that a vesicular arbuscular endomycorrhizal infection results in the synthesis of new soluble proteins in different host-fungus combinations and enhances production of some already present in uninfected roots. This is in agreement with previous reports of modi­fications in protein metabolism in tobacco and soybean (Dumas et aI., 1989, 1990; Pacovsky, 1989; Wyss et aI., 1991). Moreover, this study comparing molecular modifications oc­curring in pea mutants that are resistant to endomycorrhizal fungi and! or Rhizobium provides further evidence that specific proteins are produced only when roots successfully interact with vesicular arbuscular fungi, some of which are probably due to modifications in host protein synthesis and others to changes in expression of the fungal genome.

Acknowledgement

This work was partly supported by a BRIDGE (EEe) program grant (L.S.).

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