Alteration of enod40 Expression Modifies Medicago truncatula Root Nodule Development Induced by Sinorhizobium meliloti

Correspondence to: Martin Crespi, Martin.Crespi{at}isv.cnrs-gif.fr (E-mail), 33-1-69823695 (fax)

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Molecular mechanisms involved in the control of root nodule organogenesis in the plant host are poorly understood. One of the nodulin genes associated with the earliest phases of this developmental program is enod40. We show here that transgenic Medicago truncatula plants overexpressing enod40 exhibit accelerated nodulation induced by Sinorhizobium meliloti. This resulted from increased initiation of primordia, which was accompanied by a proliferation response of the region close to the root tip and enhanced root length. The root cortex of the enod40-transformed plants showed increased sensitivity to nodulation signals. T₁ and T₂ descendants of two transgenic lines with reduced amounts of enod40 transcripts (probably from cosuppression) formed only a few and modified nodulelike structures. Our results suggest that induction of enod40 is a limiting step in primordium formation, and its function is required for appropriate nodule development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The symbiotic interaction between leguminous plants and rhizobia results in the development of the nodule, a novel organ on plant roots. Here, bacteria provide fixed nitrogen to the host. Briefly, the microsymbionts attach to and enter the root hair cells while the plant forms a tubelike infection thread through which the bacteria move into the root cortex. Simultaneously, cortical cells are induced to divide, and the infection threads invade these dividing cells, forming the nodule primordium. This primordium differentiates into a mature nodule in which rhizobia are converted into bacteroids and start to fix nitrogen (Mylona et al. 1995 ; Cohn et al. 1998 ).

Nodule development is controlled by an exchange of signals between the two partners. Rhizobia induce nodule morphogenesis on the plant by producing nodulation signals (Nod factors; Denarie and Cullimore 1993 ; Schultze and Kondorosi 1998 ). These Nod signals trigger the earliest stages of nodule development, including root hair deformation and curling, cortical cell division, and the expression of several early nodulin genes, such as enod12 and enod40, in the growing root hair zone of alfalfa (Crespi et al. 1994 ; Journet et al. 1994 ; Bauer et al. 1996 ; Fang and Hirsch 1998 ). Thus, Nod factors act as primary morphogenetic signals for nodulation, although other plant factors also are required for the initiation of nodule morphogenesis—including plant hormones (Libbenga et al. 1973 ; Hirsch and Fang 1994 ), the stele factor uridine (Smit et al. 1995 ), the metabolic status of the plant (high carbon and low nitrogen; Bauer et al. 1996 ), and the action of the phytohormone ethylene (Heidstra et al. 1997 ; Penmetsa and Cook 1997 ). The latter hormone may be a potent inhibitor of nodulation, blocking the formation of nodule primordia (Lee and LaRue 1992 ). Moreover, it has been implicated as a second signal in the inhibition of nodulation by both light and nitrate (Ligero et al. 1991 ; Lee and LaRue 1992 ). Inhibitors of ethylene biosynthesis have been reported to increase nodule formation on alfalfa and vetch (Peters and Crist-Estes 1989 ; Zaat et al. 1989 ) and to modify positioning of cortical cell division in roots (Heidstra et al. 1997 ). Recently, a role for ethylene in nodulation was clearly demonstrated by the isolation of a supernodulating Medicago truncatula mutant that was insensitive to ethylene (Penmetsa and Cook 1997 ).

Molecular mechanisms involved in the control of nodule organogenesis in the plant host are poorly understood and have been deduced mainly from studies characterizing Nod factor action on plant gene expression and morphogenesis (Mylona et al. 1995 ; Cohn et al. 1998 ; Schultze and Kondorosi 1998 ). One of the earliest nodulin genes associated with the nodule developmental program is enod40. enod40 genes have been cloned from a number of legumes (Mylona et al. 1995 ) as well as from the nonlegume tobacco (van de Sande et al. 1996 ). In response to bacterial inoculation of roots, enod40 transcripts are detected first in the root pericycle opposite to the protoxylem pole and then in the dividing cortical cells and in all differentiating cells of the growing nodule primordia (Kouchi and Hata 1993 ; Yang et al. 1993 ; Asad et al. 1994 ; Crespi et al. 1994 ). By using promoter–Escherichia coli uidA fusions and in situ hybridization, expression of enod40 in the outer cortex preceding cortical cell division also has been reported in alfalfa (Fang and Hirsch 1998 ). In different legumes, its expression has also been detected at low levels in adventitious and lateral roots (Papadopoulou et al. 1996 ), in stem cells adjacent to the secondary phloem, and at the margins of young leaf primordia (Asad et al. 1994 ). These data indicate that enod40 may play roles in several plant organogenetic processes, although the strongest induction occurs during nodule initiation.

In Medicago spp, enod40 is also induced by purified Nod factors at concentrations required for the induction of cortical cell division as well as by cytokinins (Crespi et al. 1994 ; Fang and Hirsch 1998 ). Recently, we reported that under nitrogen-limiting conditions, overexpression of this gene resulted in a significant increase of cortical cell divisions in M. truncatula roots. This effect was observed primarily in the upper root region (Charon et al. 1997 ). Furthermore, bombardment of nitrogen-starved M. sativa roots with a cauliflower mosaic virus 35S promoter–enod40 construct induced cortical cell divisions as well as the expression of enod12A. These results suggest that enod40 action may play a role in initiating nodule morphogenesis, most likely via the modulation of hormonal imbalances in cortical cells.

In this report, we demonstrate that the overexpression of enod40 in M. truncatula plants infected by Sinorhizobium meliloti resulted in fast nodulation of these transgenic plants. Nodulation was accompanied by the proliferation of cortical cells in the root tip region and an increase in root length. At the same time, no significant differences in the final nodule numbers were found in comparison with control plants. Inoculation of enod40-transformed plants with S. meliloti mutants and treatment with purified Nod factors showed that the root cortex of these plants exhibited increased sensitivity to the Nod signals. Of 10 transgenic lines, 2 showed a reduced and perturbed nodulation phenotype, whereas enod40 transcripts could not be detected in their roots, possibly as a result of cosuppression (Elmayan and Vaucheret 1996 ). Our results indicate that induction of enod40 is required for nodule development.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Transgenic M. truncatula Plants Overexpressing enod40 Exhibit Accelerated Nodulation Kinetics
Several transgenic M. truncatula plants overexpressing the Mtenod40 gene from the cauliflower mosaic virus 35S promoter (termed Se40 plants) were previously obtained (Charon et al. 1997 ). Pools (20 to 40 plants) of independent transgenic T₂ lines (mainly from Se40 lines 1, 3, and 10) grown in vermiculite under nitrogen limiting conditions were inoculated with wild-type Sm41 (strain 41 of S. meliloti). Two phenotypes resulting from bacterial inoculation were detected for the enod40-transformed plants: accelerated nodulation kinetics and enhanced primary root growth (Figure 1). At 18 days postinfection (DPI), Se40 plants exhibited more nodules than did the control transgenic plants, and the nodules appeared in a cluster close to the root tip (see below). However, the enod40 and control transgenic plants reached approximately the same maximum number of nodules at 32 DPI (Figure 1A). The significance of these data was confirmed by using Student's t test analysis for days 18 (P < 0.01) and 23 (P < 0.05). At day 32, the differences were no longer significant. During symbiosis, the mean root length of the enod40-transformed plants was also slightly, albeit significantly, stimulated during the first 6 DPI (Figure 1B; P < 0.05). This phenotype was due to the symbiotic interaction because without bacterial inoculation no significant difference in root length was observed in the presence or absence of combined nitrogen in the medium (Charon et al. 1997 ; Figure 1B, inset).

View larger version (20K): [in this window] [in a new window]   Figure 1. Nodulation of M. truncatula Transgenic Plants Carrying the 35S Promoter–Mtenod40 Construct. (A) The number of nodules was counted on control and T2 Se40 plants at different days after infection with Sm41 (20 to 40 plants were used per time point; values are mean ±SE). Asterisks indicate significant differences (P < 0.05). At 32 DPI, differences were no longer significant. At 18 DPI, five independent experiments were performed, whereas three were performed for the remainder of the time points. (B) Root lengths in centimeters of control and Se40 plants from germination (-9 DPI) (mean ±SD). The triangle indicates inoculation with S. meliloti Sm41 (0 DPI). Note that roots grew similarly the first 9 days before infection. Asterisks indicate significant differences (P < 0.05). Three independent experiments were performed. The inset shows that no differences in root length (in centimeters) were detected in uninoculated plants (-N, without nitrogen). Note that the effect of enod40 on root growth occurred before (or at least simultaneously with) the appearance of visible nodules on the roots.

Microscopic analysis of infected roots and developing nodules was conducted by using an Sm41 derivative strain constitutively expressing lacZ so that bacteria would be readily detectable. Eight days after infection of roots from enod40 transformants, ß-galactosidase (ß-gal) staining revealed that bacteria were mainly present near the growing root hair zone and had started to enter the plant root via infection threads (Figure 2A and Figure 2B). A considerable increase in the number of infection threads reaching the inner cortex was detected for the enod40 transformants at early time points. Between 5 and 40 infection threads were detected in the enod40-transformed plants within the first centimeter of the root, whereas <5 infection threads were counted in all control plants at the same time point. In both cases, at least 20 plants were analyzed. Hence, Se40 roots showed more persistent rhizobial infections, which gave rise to the formation of nodules in clusters within the root tip zone (Figure 2B), than did roots from transgenic control plants. At the same stage of development (determined by the relative growth of infection threads associated), the primordia were larger in Se40 roots (cf. Figure 2C and Figure 2D). Transverse and longitudinal sections of these roots confirmed the presence of increased divisions in the whole cortex, with cells dividing both anticlinally and periclinally (Figure 2E to 2G). In the root pericycle, however, no increase in the number of cell divisions could be observed. During a later stage of nodulation, bacteria were detected from the invasion zone up to the central symbiotic zone (Hirsch 1992 ) in both enod40-transformed and transgenic control plants (Figure 2H to 2J). However, a region of extensive cortical cell divisions enveloped the developing nodules of the Se40 plants (Figure 2I). This proliferation phenotype was not detected for roots of control plants. Nonetheless, some nodules appearing on the upper part of roots from enod40 transformants were similar to those observed in transgenic control plants (data not shown).

View larger version (147K): [in this window] [in a new window]   Figure 2. Microscopic Analyses of Infected Roots at Different Stages of Nodule Morphogenesis. (A) and (B) Root tips of control (A) and Se40 (B) plants 10 days after infection with Rm41 (pXLGD4) constitutively expressing lacZ. Increased numbers of infection threads (arrowheads) and developed nodule primordia were detected in the Se40 plants, whereas few bacteria were observed in the control roots. ß-Gal staining was performed at the same times, and the differences could not be attributed to inefficient enzymatic reactions because some control plants occasionally showed stronger blue signals. Bars = 80 µm. (C) and (D) Representative initiation events in the growing root hair region of a control root (C) and an Se40 root (D) at a similar stage of development (determined by the relative growth of infection thread associated with the primordium). The stars indicate the initial cortical cell divisions in response to bacteria, and arrowheads indicate an infection thread. Note the size difference of the primordia in the two roots. Bars = 20 µm. (E) and (F) Transverse sections (8 µm) through a root tip region (~0.5 cm from the apex) at 10 DPI from control (E) and Se40 (F) plants, respectively. Note the increased cell proliferations in the cortex of Se40 roots compared with the control. Bars = 50 µm. (G) Longitudinal section (10 µm) of a root tip region from a transgenic enod40 plant 10 DPI. Note the extensive proliferation of the inner cortical cell layers. Bar = 20 µm. (H) Young nodule of a control root 15 DPI with Sm41 (pXLGD4) constitutively expressing lacZ. Bacteria were detected from the invasion zone up to the central symbiotic zone. Bar = 80 µm. (I) Young nodules and developed nodule primordia on an Se40 root at 12 DPI with the same Sm41 derivative strain as given in (H). No difference in bacterial localization was detected; however, the nodules are enveloped by proliferating cortical cells. Bar = 70 µm. (J) Longitudinal section (10 µm) of a root tip region from an Se40 plant 15 DPI with the same Sm41 derivative strain as given in (H). Note the presence of clustered nodules surrounded by extensive dividing cortical cells. Bar = 100 µm.

Molecular analysis of Se40 plants revealed that their roots and leaves showed increased amounts of enod40 transcripts resulting from constitutive expression of the transgene (Charon et al. 1997 ; Figure 3A). Moreover, expression of two other nodulin genes in the root tip region was analyzed by reverse transcription–polymerase chain reaction (Figure 3B). The expression of the early nodulin gene Mtenod12 (Journet et al. 1994 ) was clearly enhanced in roots from enod40-transformed plants at 6 DPI (Figure 3B). Occasionally, a very reduced basal expression at 0 DPI was detected in control plants, a possible consequence of the presence of lateral root primordia in our samples. On the other hand, no differences could be observed in the expression of MtCA1 (Coba de la Pena et al. 1997 ) in roots from control and enod40-transformed plants. Quantitative analysis of three independent experiments (data not shown) indicated that the acceleration of nodulation kinetics was correlated with an enhancement of enod12 expression.

View larger version (49K): [in this window] [in a new window]   Figure 3. Nodulin Gene Expression in Transgenic enod40 Plants. (A) RNA gel blot analysis of enod40 expression in leaves (L) and roots (R) of wild-type (control) and enod40-transformed (Se40) plants. (B) Representative semiquantitative reverse transcription–polymerase chain reaction analysis of enod12 and CA1 expression in the root region close to the root tip of control and transgenic plants during nodulation. In all cases, Msc27 was used as a constitutive control. Three independent experiments (from three RNA preparations for each time point) were performed, and all showed similar results.

Thus, the S. meliloti–inoculated Se40 plants exhibited specific proliferation responses associated with enod40 expression: increased root growth and accelerated nodulation accompanied by extensive cortical cell divisions in the region close to the root tip.

Proliferation Responses Induced on Transgenic Plants Overexpressing enod40 Depend on Nod Factor Signaling
To investigate the formation of nodules blocked at various developmental stages, enod40 and transgenic control roots were inoculated with several S. meliloti mutants. We used (1) a Bac^- mutant able to infect the nodule but unable to differentiate into bacteroids; (2) an EPS^- (for exopolysaccharide) mutant not capable of invading nodules via infection threads; and (3) a nonnodulating NodC^- mutant (Bauer et al. 1997 ). Twenty-three days after infection, microscopic analyses of roots infected by the NodC^- mutant revealed no significant alteration of the root structure compared with that of the uninfected Se40 plants. An increase in the number of cortical cell divisions was observed both in NodC^- mutant–infected and uninfected Se40 roots compared with control roots, as was shown previously for the uninfected Se40 plants (Charon et al. 1997 ).

By contrast, enod40 transformants infected with either EPS^- or Bac^- mutants exhibited accelerated nodulation kinetics. A few empty nodules appeared on the enod40-transformed roots at 23 DPI when the EPS^- mutant was present, whereas they first appeared 1 week later on control plants. Clustered nodule initiation and extensive proliferation of cortical cells were observed in the root tip region of Se40 roots infected with the EPS^- mutant (data not shown). When the Se40 roots were infected with the Bac^- mutant, the extent of nodulation detected at 23 DPI was threefold greater than in the roots of transgenic control plants infected with the Bac^- mutant. The Bac^- mutant–induced nodules appearing on Se40 roots were also enveloped in a proliferating region (data not shown). Moreover, the Se40 roots also were more elongated (mean root length was 4.4 ± 0.8 cm and 5.6 ± 0.8 cm for control and Se40 roots, respectively, a statistically significant difference [P < 0.05]). These results further suggest that both proliferation responses in the Se40 plants are related to Nod factor action.

To confirm these results, the effect of purified Nod factors was tested on control and Se40 plants. Treatment with NodSmIV(S) purified from S. meliloti at 10^-7 M revealed root responses in the growing root hair zone, including root hair deformation and curling as well as cortical cell divisions. Detailed microscopic analysis of this zone revealed three types of dividing cell foci, which could be distinguished by the location, orientation of divisions, and the number of root hairs present on the corresponding epidermis (Figure 4): (1) dividing cells in the pericycle and cortex (putative lateral root primordia; Figure 4A), (2) anticlinally dividing cells in all cell layers of the cortex and high quantities of root hairs (Figure 4B), or (3) clusters of cells in the inner cortex that divided both anticlinally and periclinally (Figure 4C). These latter foci may correspond to putative Nod factor–induced nodule primordia. After treatment of enod40 and transgenic control roots with the Nod factor at different concentrations, plants overexpressing enod40 demonstrated a significant increase in the number of the type 3 foci within the first centimeter of the root region close to the tip (Figure 4D). No differences were found in the number of the two other focus types (data not shown).

View larger version (75K): [in this window] [in a new window]   Figure 4. Treatment of Root Tissues with Purified Nod Factors. (A) to (C) Root region of an Se40 plant 2 days after NodSmIV(S) (10-7 M) treatment, showing the following: (A) a lateral root primordium with dividing cortical cells and divisions in the pericycle (type 1 foci; bar = 20 µm); (B) anticlinally dividing cells in all layers of the root cortex, with high quantity of root hairs (type 2 foci; bar = 50 µm); and (C) anticlinally and periclinally dividing cells in the inner root cortex (type 3 foci; bar = 40 µm). (D) Number of type 3 foci within the first centimeters of the root region close to the tip of plants 2 days after treatment with different concentrations of NodSmIV(S), 10-7 M N,N',N'',N'''-tetraacetylchitotetraose, and water (mean ±SE). Asterisks indicate statistically significant differences (P < 0.05). Chito, chitotetraose.

These results suggest that the different responses observed after S. meliloti infection of Se40 roots in the root tip region are due to a synergism between the Nod factor and enod40 action.

Nitrogen and Ethylene Control enod40 Action
A limited supply of nitrogen is a prerequisite for nodule initiation and development. To test whether nitrogen limitation was also a prerequisite for the observed nodulation phenotype, Se40 plants were grown in media containing 1, 2, or 5 mM potassium nitrate and inoculated with S. meliloti 8 days after germination. Eighteen days after inoculation, the nodules on these plants were counted. Figure 5A shows that enod40-mediated acceleration of nodulation was repressed significantly by potassium nitrate concentrations as low as 1 mM (50% reduction in nodule number). To test whether enod40 action could be regulated by nitrogen once nodules were formed, at 10 DPI, potassium nitrate was applied at a concentration of 20 mM to transgenic plants harboring developing nodules. Counting nodules at 0, 4, 8, and 16 days after nitrate treatment indicated that the supply of potassium nitrate blocked nodulation both in control and enod40 transformants (Figure 5B; data not shown). Plants that were maintained 8 or 16 days after the nitrogen treatment showed an increase in nodule number (Figure 5B), probably because the plant metabolized the added nitrogen.

View larger version (19K): [in this window] [in a new window]   Figure 5. Nitrogen and Ethylene Control the Action of enod40. (A) Number of nodules on control and Se40 roots 18 DPI. Plants were grown on nitrogen-free medium or on medium supplemented with potassium nitrate at a final concentration of 0, 1, 2, or 5 mM. Twenty to 40 plants were used in at least two independent experiments. (B) Plants harboring developing nodules were treated at 10 DPI with a 20 mM potassium nitrate solution. Nodule numbers in independent plant pools (20 plants per time point) were counted 0, 4, 8, or 16 days after treatment (T'). (C) Mean root length (in centimeters) of plants grown for 5 days on agar plates in a medium containing 0.25 mM potassium nitrate supplemented with different concentrations of ACC. Root length is expressed as the mean ±SD. The asterisk indicates the only slight albeit statistically significant difference (P < 0.01). (D) Number of nodules on plant roots infected with Sm41 or supplemented with ACC and AVG at 15 and 23 DPI. In (A), (B), and (D), nodule number is expressed as the mean ±SE.

As mentioned above, Se40 plants showed a strong proliferation of cortical cells (Figure 2F), which may indicate a loosening of ethylene control (Heidstra et al. 1997 ) in the enod40 transformants. 1-Aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, is able to inhibit several responses in M. truncatula plants, including root growth, hypocotyl elongation, and to induce the formation of a hypocotyl hook (Penmetsa and Cook 1997 ). Hence, control and Se40 seedlings grown on low-nitrogen agar medium were treated with ACC for 5 days. No considerable differences in primary root length (or in the other responses) were observed in the Se40 plants compared with control plants (Figure 5C; data not shown), except for a slight but statistically significant (P < 0.01) increase in root length at 0.01 mM ACC. However, the degree of inhibition of root elongation at different ACC concentrations indicated that both sets of plants were sensitive to ethylene. In addition, plants were treated with ACC or aminoethoxyvinylglycine (AVG; an inhibitor of ACC synthase) with or without concomitant S. meliloti inoculation. A marked reduction in nodule number at 15 and 23 DPI was found after treatment with either 10^-5 M (Figure 5D) or 10^-7 M ACC (data not shown), confirming that in M. truncatula plants, ethylene negatively regulates nodulation (Penmetsa and Cook 1997 ). Despite the repression of nodule formation by ACC in Se40 plants, their differential nodulation kinetics were maintained at both concentrations. When the ACC synthase inhibitor AVG (10^-7 M) was applied to roots of bacterially infected plants, the control plants treated with AVG showed enhanced nodulation similar to that of enod40 transformants (Figure 5D). However, microscopic analyses of these control roots revealed only a few cases of extensive cortical cell divisions in the infected region, unlike our observations in the Se40 plants (data not shown).

These results indicate that in Se40 plants, the control of nodulation by ethylene or nitrogen or a plant's sensitivity to ACC was not altered markedly. However, the enod40 action could be partially mimicked by treatment of infected roots with an ethylene inhibitor.

Two Transgenic Lines with Suppressed Levels of enod40 Transcripts Exhibit Reduced Nodulation Capacity
Of 10 transgenic lines analyzed in detail, the Se40 lines 2 and 5 showed a marked reduction of enod40 transcripts in their leaves (Charon et al. 1997 ; data not shown). This might be due to a cosuppression phenomenon induced by the transgene (Elmayan and Vaucheret 1996 ). To determine the effect of the reduced amounts of enod40 transcripts, we analyzed several T₁ and T₂ seedlings from the progeny of these plants for their nodulation behavior and enod40 expression. Under aeroponic nodulation conditions, between 40 and >130 nodules per control plant were detected at 32 DPI (with a mean of ~100 nodules per plant; Figure 6A). For the T₁ and T₂ plants of both enod40 transgenic lines, a striking difference in the frequency of nodule number was detected, with certain plants harboring <10 nodules (Figure 6B to 6D). More than 20 individual plants were analyzed for enod40 expression in roots, nodules, and leaves and for nodulation behavior. In both T₁ and T₂ generations, we found a correlation between the arrest of nodule development and the strongly reduced amounts of enod40 transcripts (analysis of 11 plants from Se40 line 2 are shown in Figure 7A). Moreover, in 20% of the plants, expression of the transgene was still detectable in leaves but not in nodules (Figure 7B). This suggests that cosuppression was triggered by the nodulation process, during which the endogenous transcript accumulates to high levels.

View larger version (28K): [in this window] [in a new window]   Figure 6. Nodulation Frequencies of Selected Se40 Lines. Distribution of plants according to their nodule number at 32 DPI under aeroponic conditions. (A) Wild type (WT). (B) T1 generation of the transgenic enod40 Se40 line 2 (Se40(2) T1). (C) T2 generation of transgenic enod40 Se40 line 2 (Se40(2) T2). (D) T2 generation of the transgenic enod40 Se40 line 5 (Se40(5) T2). Note the shift in nodule number between control and Se40 distributions.

View larger version (29K): [in this window] [in a new window]   Figure 7. Nodule Number and enod40 Expression Analysis in Selected Se40 Lines. (A) Correlation plot between nodule number at 32 DPI and levels of enod40 expression in nodules from individual descendants of transgenic Se40 lines 2 and 5. Note the two distinct populations. Squared points represent control plants. A.U., arbitrary units. (B) RNA gel blot analysis of enod40 expression in leaves (L) and nodules (N) of wild-type plants (WT) and a transgenic enod40 plant of the Se40 line 2 (Se40) showing reduced nodulation. Msc27 was used as a loading control.

The reduction in the nodulation capacity of these plants was observed by using different systems (aeroponic or vermiculite growth conditions) for the nodulation assay (data not shown). At 32 DPI, a considerable proportion of the T₂ progeny of Se40 line 5 grown in vermiculite did not contain nodules (Figure 8A) and showed abnormal distribution of nodulation in comparison with control transgenic plants (Figure 8B and Figure 8C). Moreover, the progeny of a Nod^- plant yielded plants exhibiting variable nodulation capacities (Figure 8D). No nod^- allele segregating independently of the transgene in the T₁ or T₂ generation of these plants could be detected (data not shown). The segregation as well as the correlation between the lack of detectable enod40 expression and the strongly reduced nodulation capacity suggests that cosuppression of the endogenous enod40 gene is responsible for the arrest of nodule development.

View larger version (28K): [in this window] [in a new window]   Figure 8. Percentage and Frequency of Nodulation of T2 and T3 Descendants from the Transgenic Se40 Line 5. (A) T2 descendants of transgenic Se40 line 5 (Se40(5) T2) were inoculated with S. meliloti in Magenta jars containing vermiculite. At different times after infection, the percentage of nodulated plants was scored on independent plant pools. At 32 DPI, 43% of the transgenic plants did not contain any visible nodules on their roots. (B) and (C) Representative distribution of plants according to their nodule number at 32 DPI. Whereas almost all of the transgenic control plants (48 out of 50) showed at least one nodule (B), markedly less nodulation was observed for the transgenic Se40 line 5 T2 plants (Se40(5) T2) (C). Eight of 18 plants analyzed did not contain any nodules. (D) T3 descendants (42 plants) from one Nod- plant depicted in (C) (Se40(5) T3) were nodulated. At 32 DPI, Nod- and Nod+ phenotypes could be detected clearly for these plants. In these conditions of nodulation (Magenta jars containing vermiculite), only a few nodules per plant appeared (one to five nodules) in contrast to the plant grown under aeroponic conditions.

In these enod40-cosuppressed plants, nodules were small and perturbed in their development. Histological analysis of arrested nodules showed the absence of meristem organization and nodule zonation (cf. Figure 9A and Figure 9B). Moreover, although under certain conditions invasion and bacteroid differentiation took place in a few central cells of the small nodules, strong accumulation of fluorescent products indicated the senescence of a large region (Figure 9C). In addition, root tissues near the mature nodules contained cortical cells that were larger than those that had undergone division in the infected regions of control roots (data not shown).

View larger version (120K): [in this window] [in a new window]   Figure 9. Modified Nodules at 32 DPI Induced on Roots of Se40 Lines 2 and 5. (A) Transverse sections (10 µm) of a wild-type nodule showing the characteristic indeterminate nodule zonation (I, meristem; II, infection zone; III, fixation zone). Bar = 90 µm. (B) Transverse sections (10 µm) of a nodule from a T1 transgenic Se40 line 2 plant, in which no well-organized tissues can be recognized. Bar = 80 µm. (C) Transverse sections (10 µm) of the same nodule as (B) but observed under fluorescence, highlighting the presence of a large senescent region in the arrested nodule. Bar = 70 µm.

These results suggest that expression of enod40 is an essential step in nodule development as well as in the proliferation of root cortical cells induced by infection with the bacterial symbiotic partner.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

In this study, we show that altered levels of enod40 expression considerably affect nodule induction by S. meliloti. During this symbiotic interaction, overexpression of enod40 resulted in accelerated nodulation and enhanced root growth of transgenic plants. Moreover, those plants in which silencing of enod40 gene expression (possibly mediated by cosuppression) was detected exhibited reduced nodulation capacity, and the nodules were arrested in their development. Hence, either overexpression or underexpression of enod40 resulted in marked alterations of the responses of root tissues to the symbiotic bacterial partner. This indicates that expression of enod40 in roots is a limiting step required for the proper development and organization of the nodule tissues.

The early nodulin gene enod40 is expressed at the onset of nodulation and in the differentiating cells of the nodule in alfalfa and several other legumes (Kouchi and Hata 1993 ; Yang et al. 1993 ; Crespi et al. 1994 ; Fang and Hirsch 1998 ; reviewed in Schultze and Kondorosi 1998 ). In our experiments, the action of enod40 was detected in the region, close to the root tip, that is sensitive to Nod factors (Schultze and Kondorosi 1998 ), whereas other root regions did not show major alterations in their division patterns during the symbiotic interaction. Accumulation of the enod40 transcript in the root pericycle occurs very soon (3 to 12 hr) after treatment with the Nod factor, preceding cortical cell division (Mylona et al. 1995 ; Vijn et al. 1995 ; Minami et al. 1996 ). However, enod40 induction also can be detected by using chitin pentamers (unable to induce cortical cell division) in soybean roots (Minami et al. 1996 ). This result indicates that expression of enod40 in the pericycle is not sufficient to promote cell division in the cortex and supports the notion that Nod factors are likely to act as signals in both tissues.

The enod40 genes (Cohn et al. 1998 ; Fang and Hirsch 1998 ) encode RNAs of ~0.7 kb that contain only short open reading frames (ORFs). Modeling predicts that enod40 RNA sequences have the tendency to form particularly stable secondary structures. This property is shared with several biologically active RNAs (Crespi et al. 1994 ). On the other hand, a very small ORF corresponding to 10 to 13 amino acids in the 5' end of the transcripts is common among these genes and has been proposed to be the active gene product (Vijn et al. 1995 ; van de Sande et al. 1996 ). Recently, we have demonstrated that several small ORFs of Mtenod40 were translated when fused to a reporter gene (C. Sousa, C. Johansson, C. Charon, H. Manyani, C. Sautter, A. Kondorosi, and M. Crespi, manuscript in preparation). Thus, further work is necessary to elucidate the molecular mechanism of enod40 action in plant tissues.

The nodulation phenotype induced by overexpression of enod40 is not related to supernodulation (Caetano-Anolles and Gresshoff 1991 ; Penmetsa and Cook 1997 ; Szczyglowski et al. 1998 ). Although at early time points an increase in nodule number was detected in the transgenic enod40 plants, after 1 month no significant differences in the total nodule number were found. Thus, overexpression of enod40 modified the early stages of nodulation and infection. Afterward, the autoregulation of nodulation most likely controls the total number of nodules in both control and Se40 plants (Caetano-Anolles and Gresshoff 1991 ; Mylona et al. 1995 ). Moreover, nodules obtained from Se40 plants were red (due to the presence of leghemoglobin) and fully developed, in contrast to certain supernodulator mutants in which the very high number of nodule initiations resulted in delayed or abnormal nodulation (Caetano-Anolles and Gresshoff 1991 ). Mutants affected in nitrate sensitivity were also affected in nodulation; however, the dose–response curve for the effect of nitrogen on nodulation suggests that the transgenic enod40 plants are even more sensitive to nitrate action than are control plants.

The effect of nitrate and the positioning of the nodules were proposed to be mediated by ethylene (Ligero et al. 1991 ; Lee and LaRue 1992 ; Heidstra et al. 1997 ). enod40 induction in the cortex may be a determinant of nodule primordium formation because enod40 overexpression resulted in the proliferation of cortical cells and an increased number of successful infections by S. meliloti, leading to the formation of nodule clusters with extensive cortical cell divisions in the root tip region. This increased infection response is related to the skl-conferred (for sickle) phenotype that has been linked to ethylene sensitivity (Penmetsa and Cook 1997 ), although in Se40 plants we could not detect similar changes in root growth or in nodulation responses to ethylene. Nevertheless, treatment with ethylene inhibitors partially mimicked enod40 overexpression, suggesting that the interaction between enod40 and ethylene may be complex. Further studies are required to analyze the relation of gene function with the responses to ethylene or other hormones.

In our study, a putative cosuppression phenomenon yielded plants showing arrested nodule development. Moreover, the reduction in nodule formation capacity that we detected suggests that nodule initiation was also affected in the suppressed plants. Establishment of cosuppression may require triggering the expression of the endogenous gene (Baulcombe 1996 ; Elmayan and Vaucheret 1996 ), as occurred with enod40 during nodule initiation. Despite this correlative evidence (in contrast to a strict extinction of gene function by a natural or induced mutation), these data provide strong support for our notion that enod40 is required for nodule development. The observed low extent of nodule initiation might also be due to sectors in which suppression did not take place or to reversion of the suppression induced by the plant's demand for nitrogen. In fact, cosuppression has been shown to be under environmental control (Baulcombe 1996 ). Lotus japonicus mutants affected in vascular tissue and nodule development that formed determinate nodules without a persistent meristem also contained highly reduced amounts of enod40 transcripts (Szczyglowski et al. 1998 ). In these cases, however, the perturbations in nodule development might be caused by other genes affected by the mutation. In these L. japonicus mutants, nodule initiation was normal. However, two enod40 genes are present in L. japonicus (Flemetakis et al. 1998 ), and further studies are required to determine whether enod40 induction is absent in the initial dividing cortical cells of these mutant determinate nodules.

enod40 has been suggested to play different roles in nodule development, for example, in the transport of certain key compounds or more directly in the induction of cell division (Yang et al. 1993 ; Crespi et al. 1994 ; Mylona et al. 1995 ; Minami et al. 1996 ; Charon et al. 1997 ; Fang and Hirsch 1998 ). A link between enod40 action and hormone signaling has been suggested (Mylona et al. 1995 ; van de Sande et al. 1996 ; Charon et al. 1997 ). For example, ectopic overexpression of this gene affected certain hormonal responses of somatic embryos of alfalfa under in vitro culture conditions (Crespi et al. 1994 ). Moreover, in the absence of S. meliloti, a proliferation response in the upper root region of the transgenic enod40 plants grown under nitrogen-limiting conditions was detected (Charon et al. 1997 ). Here, we report that during nodule development, the region close to the root tip showed strong proliferation of cortical cells and increased infection by S. meliloti, revealing a synergism of enod40 and a Nod factor–regulated pathway in root tissues.

The action of enod40 varies depending on plant cell type or environmental conditions. Therefore, we think that the primary function of this gene is not exerted directly on triggering cell division per se but on sensitizing the cells for division. In contrast to auxin, enod40 does not have the capacity to induce cell proliferation irrespective of the plant's nitrogen status or root position. Moreover, during certain mycorrhizal symbioses, enod40 is induced in cortical cells that contain arbuscules whereas the division of those cells is not (van Rhijn et al. 1997 ). Hence, a role of enod40 in the transport of compounds into the cortex or in the modification of cell-to-cell communication to allow proper organization of the nodule primordium seems to be more likely. This may in turn affect the regulation of hormone signaling in the enod40-expressing cells, resulting in the appropriate type of cortical cell divisions required for the formation of the nodule primordium. Our work suggests that enod40 may determine which cells can undergo division.

Although our experiments did not reveal the mechanism of enod40 function inside the nodule cells, they indicate that this gene is an important determinant of nodule formation and development. Understanding how a short ORF-containing RNA provokes these effects at the cellular level may have profound implications for the study of plant development and also may reveal a novel mechanism of regulation involved in cell differentiation in eukaryotes.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Material and Plant Growth
Plants (Medicago truncatula cv 108R) overexpressing the early nodulin enod40 gene have been described previously (Charon et al. 1997 ). The T₁ or T₂ progenies as well as control plants were used for our experiments. As control plants, we used wild-type 108R or transgenic plants carrying the pG3.3 vector. Seeds were sterilized by immersion for 1 hr in Inov'chlore (8.64 g/L; Inov'chem SA, Tanneries, France) and sodium dodecyl sulfate (1 g/L), followed by thorough washing overnight with sterile water. Germinated seedlings (1 day old) were transferred to Magenta jars or squared Petri dishes containing vermiculite embedded in nitrogen-free Gibson medium (as given in Bauer et al. 1996 ). For growth on Petri dishes, moist vermiculite was added to fill half of a plate that was placed almost vertically to allow root and nodule observations for different time periods. One week later, roots were inoculated with Sinorhizobium meliloti or treated with Nod factors (see below). Growth of seedlings in vermiculite was required for the nodulation assays because roots of M. truncatula cv 108R do not grow properly for long time periods (i.e., 10 to 30 days) on agar plates. In contrast, short-term (i.e., 5 days) ACC treatments were performed by transferring the germinating seedlings to new agar plates containing ACC at different concentrations. Plants were grown in a growth chamber at 24°C under a 16-hr light period.

Nodulation Assays
S. meliloti strains grown overnight (as described in Coba de la Pena et al. 1997 ) were centrifuged and resuspended at an OD₆₀₀ of 0.4 in 10 mM MgSO₄. Bacterial inoculum (300 µL) was applied to each seedling. For nodulation studies and combined treatments with ACC and AVG, the wild-type S. meliloti strain 41 (Sm41) was used. Early steps of nodulation were monitored with an Sm41 strain carrying the pXLGD4 plasmid with a -ala::lacZ fusion (Leong et al. 1985 ). Wild-type S. meliloti strain 1021 (Sm1021) and its NodC^- (EK1652), EPS^- (Sm0540), and Bac^- (Sm8368) mutants were used for nodulation experiments (Bauer et al. 1997 ). No differences were detected between the nodulation behavior of strains Sm1021 and Sm41. On different days after inoculation or treatments, plant roots were carefully washed to remove the vermiculite, nodules were counted, and the primary root length of each plant was measured from the root tip to the hypocotyl. For each condition, 20 to 40 plants were used in at least three independent experiments.

ACC and AVG treatments combined with bacterial inoculation were performed by applying to a 1-week-old seedling an ACC or AVG solution (300 µL at 10^-5 or 10^-7 M) with or without the rhizobial inoculum. Nodules were counted at days 15 and 23, as mentioned above. For each condition, 10 to 20 plants were used and at least two independent experiments were performed.

For studying nitrogen control of nodulation, two conditions were used. In the first, plants were grown in Magenta jars containing vermiculite embedded in nitrogen-free Gibson medium supplemented with different concentrations of potassium nitrate (1 to 5 mM). Eighteen days after inoculation with Sm41, nodules were counted. Twenty to 40 plants were used in at least two independent experiments. In the second, plants were grown in Petri dishes half filled with vermiculite (containing 10 mL of nitrogen-free medium); 10 days after infection with S. meliloti, the roots were treated with 300 µL of a 20 mM solution of potassium nitrate. At days 1, 4, 8, and 16 after this treatment, roots of the independently treated pools were washed, and the number of nodules was counted. Ten to 20 plants were used in at least two independent experiments for each time point.

For studying the effect of enod40 cosuppression, nodulation behavior of the progeny of transgenic Se40 lines 2 and 5 was analyzed under aeroponic conditions (Crespi et al. 1994 ) to increase plant nodulation. Plants were kept for 2 weeks under nitrogen deprivation conditions and then infected with S. meliloti Sm41. Nodules from 15 to 30 plants were counted in at least two independent experiments for each progeny line. The nodulation capacity of T₂ and T₃ progenies of the transgenic Se40 line 5 was tested in vermiculite under normal growth conditions in Magenta jars (as given above).

Root Growth Assays
For Nod factor treatment, purified NodSm-IV (C16:2, S) (Schultze et al. 1992 ) was used at concentrations of 10^-7 to 10^-10 M N,N',N'',N'''-tetraacetylchitotetraose (Sigma) at 10^-7 M, and a water control. Nod factor solutions (300 µL) were applied around the top of the root of each seedling grown in vermiculite in Petri dishes. Two days later, roots were washed and fixed in FAA solution (50% ethanol, 5% glacial acetic acid, 10% formaldehyde, and 35% water) as described previously (McKhann and Hirsch 1993 ). The region of the root tips on each seedling was screened for cell division under the microscope (see below). For each condition, 10 to 20 plants were used in at least two independent experiments.

Nodulin Gene Expression Analyses
To test for the presence of enod40 transcripts in control and transgenic plants, total RNA was prepared by using the guanidium chloride method (Logemann et al. 1987 ) and used for RNA gel blot analysis (Charon et al. 1997 ). Msc27 served as an RNA loading control (Crespi et al. 1994 ). Semiquantitative reverse transcription–polymerase chain reaction studies were performed with total RNA (prepared as above) from the lower 1.5 cm of the roots from 40 plants, excluding the root tips (1 mm) (Crespi et al. 1994 ). MsCA1 (Coba de la Pena et al. 1997 ) and Msenod12A (Bauer et al. 1996 ) probes were prepared by using the digoxigenin RNA labeling kit (SP6 and T7; Boehringer Mannheim). Hybridization procedures and detection with the CDP-Star chemiluminescence substrate were performed according to the manufacturer's (Boehringer Mannheim) directions, with modifications as described in Dessaux et al. 1995 . Quantification of the hybridization signal intensities was performed by densitometric scanning of the blots using the NIH Image public-domain software (http://rsb.info.nih.gov/nih-image/). Means and standard deviations of the hybridization signal intensities were calculated from at least three independent RNA preparations for each time point.

Histological Analysis and ß-Gal Activity
Roots from treated M. truncatula seedlings were harvested, immediately fixed in FAA as described by Charon et al. 1997 , cleared for 15 min in commercial bleach (1.75% active chlorine), and analyzed microscopically. The first centimeter of each root was screened under eightfold magnification and Nomarski optics to search for dividing cortical cell clusters.

Prefixation and staining with ß-gal were performed according to Bauer et al. 1997 and using 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside or 5-bromo-6-chloro-3-indolyl ß-D-galactopyranoside. Tissues were cleared as described above.

For semithin sectioning, roots and nodules were fixed and embedded in Paraplast (Sherwood Medical, Athy, Ireland) according to McKhann and Hirsch 1993 before sectioning with a microtome (Leica, Rueil-Malmaison, France). Paraffin was removed from sections for observations.

Statistical Analyses
For each condition for which primary root length was measured, the mean values of the measurements were given with the standard deviation. Under each condition for which nodule number (or number of dividing cell foci) was counted, the mean value of the measurements was given with the standard deviation divided by the squared root of the number of plants. The significance of the results in all cases was tested by using Student's t test.

	FOOTNOTES

¹ Current address: Departamiento Microbiologia y Parasitologia, University of Pharmacy, 41080 Seville, Spain.

	ACKNOWLEDGMENTS

We gratefully acknowledge Michael Schultze for the Nod factor and chitotreaose solutions, Petra Bauer for the Sm41 (pXLGD4) strain, Asao Ichige for Sm8368, Alfred Pühler for Sm0540, and Eva Kondorosi for EK1652. We also thank Barry Rolfe for useful discussion. C.C. and C.S. were supported by the Ministère Français de l'Enseignement Supérieur et de la Recherche and by a postdoctoral fellowship from the European Economic Community (Training and Mobility Research Marie Curie Fellowship), respectively.

Received April 14, 1999; accepted August 2, 1999.

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