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34 Pages·2015·6.88 MB·English

Plant Physiology Preview. Published on August 18, 2015, as DOI:10.1104/pp.15.00584

1 Running head: Bacteroid differentiation in Aeschynomene legumes
2 Eric GIRAUD
3 E-mail: [email protected]
4 Phone: [phone]
5
6 Adress: Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR
7 IRD/SupAgro/INR/CIRAD/ U. Montpellier, Campus International de Baillarguet, TA A-82/J, 34398
8 Montpellier Cedex 5, France
9
10 Primary research area: Genes, Development and Evolution
11 Secondary research area : Cell Biology
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Copyright 2015 by the American Society of Plant Biologists
13 Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and
14 IRLC Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides
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16 Pierre Czernic1*, Djamel Gully2*, Fabienne Cartieaux2, Lionel Moulin2, Ibtissem Guefrachi3, Delphine
17 Patrel2, Olivier Pierre3, Joël Fardoux2, Clémence Chaintreuil2, Phuong Nguyen2, Frédéric Gressent2,
18 Corinne Da Silva4, Julie Poulain4, Patrick Wincker4, Valérie Rofidal5, Sonia Hem5, Quentin Barrière3,
19 Jean-François Arrighi2, Peter Mergaert3, Eric Giraud2.
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21 1Université de Montpellier, Place Eugène Bataillon, F-34095 Montpellier Cedex 5, France.
22 2IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR
23 IRD/SupAgro/INRA/UM2/CIRAD, Campus International de Baillarguet, TA A-82/J, 34398
24 Montpellier Cedex 5, France
25 3Institute for Integrative Biology of the Cell, UMR 9198, CNRS/Université Paris-Sud/CEA, Gif-sur-
26 Yvette, France
27 4Commissariat à l’Energie Atomique, Direction des Sciences du Vivant, Institut de Génomique,
28 Génoscope, 91000 Evry, France
29 5Laboratoire de Protéomique Fonctionnelle, Institut National de la Recherche Agronomique, Unité de
30 Recherche 1199, Montpellier, France.
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32
33 Summary: Several Aeschynomene species from the ancient Dalbergoid legume lineage independently
34 evolved a novel class of cysteine rich peptides to impose a differentiation process on their
35 endosymbionts.
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39 Footnotes:
40 * These authors contributed equally to this work
41
42 This work has been supported by the French National Research Agency (ANR-SESAM-2010-BLAN-
43 170801 and ANR-BugsInaCell-13-BSV7-0013).
44
45 Corresponding author: Eric GIRAUD
46 e-mail: [email protected]
47
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48 ABSTRACT
49 Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which
50 produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen fixing
51 symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly
52 within highly specialized symbiotic host cells. In IRLC legumes such as Medicago, the bacteroids are
53 kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the
54 bacteria in an irreversible, strongly elongated and polyploid state. Here we show that in Aeschynomene
55 legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical
56 depending on the Aeschynomene species and that these bacteroids are terminally differentiated and
57 polyploid, similarly to bacteroids in IRLC legumes. Transcriptome, in situ hybridization and proteome
58 analysis demonstrated that the symbiotic cells in the Aeschynomene nodules produce a large diversity
59 of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNAi-
60 mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results
61 support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved
62 independently from the bacteroid differentiation in the IRLC clade, is based on very similar
63 mechanisms used by IRLC legumes.
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66 INTRODUCTION
67 Legumes, thanks to their ability to develop a symbiotic interaction with nitrogen fixing bacteria,
68 collectively called rhizobia, are among the agronomically and ecologically most important plants. This
69 symbiosis results in the formation of new organs, the nodules, inside which rhizobia differentiate into
70 an endosymbiotic form, the bacteroids, able to fix atmospheric nitrogen to the benefit of the plant.
71 During this differentiation step, profound modifications of the metabolism of the rhizobia are observed
72 and this can be accompanied by a marked change in the bacterial cell shape and size (Haag et al.,
73 2013). Three distinct bacteroid morphotypes have been observed in different legume species (Oono et
74 al., 2010; Bonaldi et al., 2011; Kondorosi et al., 2013) (Supplemental Fig. S1A): i) elongated or E-
75 morphotype bacteroids described in legumes of the Inverted Repeat-Lacking Clade (IRLC) (Medicago,
76 Pisum, Vicia, …) and some Aeschynomene species such as A. afraspera, ii) enlarged, spherical
77 bacteroids (S-morphotype) encountered in some species of the Dalbergioid clade (such as
78 Aeschynomene indica, Aeschynomene evenia and Arachis hypogaea) and iii) unmodified bacteroids
79 (U-morphotype) which display a rod-shape morphology similar to free-living bacteria found in e.g.
80 Phaseoloid or Robinoid legumes (i.e. Phaseolus, Vigna, Lotus, Glycine, Sesbania). The fact that the
81 same rhizobium strain nodulating legumes of different clades can display different morphotypes is a
82 strong evidence supporting the conclusion that the host plant governs bacteroid morphotype (Sen and
83 Weaver, 1984; Mergaert et al., 2006; Bonaldi et al., 2011). The change of shape is probably the tip of
84 the iceberg of the control exerted by the plant on the bacteria physiology during symbiosis. Indeed,
85 besides their E-morphotype, the Sinorhizobium meliloti bacteroids in Medicago truncatula differ from
86 their free-living state on several aspects, they become polyploid, their membrane permeability
87 dramatically increases and they lose their reproductive capacity. Bacteroids that display such extreme
88 changes are considered as terminally differentiated because they are unable to revert to their bacterial
89 form (Mergaert et al., 2006).
90 In Medicago truncatula, a class of peptides, named NCRs (Nodule-specific Cysteine-Rich peptides)
91 plays a key role in this terminal bacteroid differentiation (Van de Velde et al., 2010). The M.
92 truncatula NCR family is composed of about 600 highly divergent genes, which are nearly all
93 exclusively expressed in nodules (Mergaert et al., 2003; Alunni et al., 2007; Young et al., 2011).
94 NCRs are similar to the defensin-type of antimicrobial peptides. The peptides contain an N-terminal
95 secretion signal, the mature peptides are usually no longer than 60 amino acids and have 4 to 6
96 conserved cysteine residues (Mergaert et al., 2003). The cleavage of the signal peptide by a nodule-
97 specific signal peptidase complex (SPC) located in the endoplasmic reticulum is required to permit the
98 trafficking of NCR peptides to the symbiosome compartment. Bacteroid and symbiosome
99 development is blocked in a M. truncatula dnf1 mutant that is affected in the SPC22 subunit of this
100 nodule-specific SPC (Van de Velde et al., 2010; Wang et al., 2010). Some NCR peptides have been
101 found to have antimicrobial activity, killing rhizobium when applied at high concentration (Van de
102 Velde et al., 2010). However, these antimicrobial peptide-like NCRs can at lower concentration induce
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103 typical features of E-morphotype bacteroids in vitro on cultured rhizobium (Van de Velde et al. 2010).
104 The NCR gene family has been identified in all investigated legume species of the IRLC clade but in
105 no other plant species outside of this clade. This raises the question about the nature of the factors used
106 by the other legumes to control bacteroid metamorphosis.
107 In the Dalbergioid legume clade, bacteroids can be of the E- or S-morphotype. For example, within the
108 Aeschynomene genus, A. afraspera has E-type bacteroids but A. indica or A. evenia have S-type
109 bacteroids (Bonaldi et al., 2011; Arrighi et al., 2012). The bacteroid morphogenesis in the
110 Aeschynomene genus is also under plant control since the Bradyrhizobium strain ORS285 transforms
111 into E-morphotype bacteroids in A. afraspera nodules and in S-morphotype bacteroids in A. indica or
112 A. evenia nodules (Bonaldi et al., 2011; Arrighi et al., 2012). The Aeschynomene symbiosis with
113 Bradyrhizobium sp. has several other particular features, including the formation of nodules on both
114 roots and stems, photosynthesis by the Bradyrhizobium symbionts which is required for optimal stem
115 nodulation (Giraud et al., 2000) and the use of a Nod factor independent nodulation pathway in some
116 Aeschynomene species including A. indica or A. evenia (Giraud et al., 2007; Arrighi et al., 2012).
117 Furthermore, in contrast to classical determinate and indeterminate nodules, aeschynomenoid nodules
118 originate from the successive divisions of only one or a few root cortical cells initially infected by an
119 infection thread-independent mechanism (Bonaldi et al., 2011). This infection mechanism has two
120 consequences: i) all the nodule primordium cells are infected and ii) all the nodule primordium cells
121 and bacteria are synchronized. Thus, during the nodule development of A. indica nodules, the
122 differentiation of bacteroids occurs simultaneously for all nodule cells and takes place between the 4th
123 and 5th day after inoculation (Bonaldi et al., 2011). The possibility to obtain sampling of homogenous
124 material at different differentiation stages and the existence of two distinct bacteroid morphotypes in
125 closely related species make the Bradyrhizobium-Aeschynomene symbiotic couples an appealing
126 model system to unravel the mechanisms of bacteroid morphotype determination in legume species
127 outside the IRLC clade.
128 In this study, we show that E- and S-morphotype bacteroids from A. afraspera and A. indica nodules
129 display typical features of terminal differentiation. Furthermore, in these two species, we reveal the
130 presence of a NCR-like peptide family which is specifically produced in the nodules and targets the
131 bacteroids. Alteration of the trafficking of these peptides by silencing the dnf1 homolog identified in A.
132 evenia correlated with the absence of bacterial differentiation indicating that the NCR-like peptides
133 identified in Aeschynomene species play a similar role to those described in Medicago. Altogether,
134 these data support the hypothesis of a convergent evolution of the host molecular mechanisms
135 governing bacteroid differentiation in legumes.
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137 RESULTS
138
139 E and S-Bacteroids in Aeschynomene Species are Terminally Differentiated
140 Previous studies revealed that Bradyrhizobium strain ORS285 displayed two distinct bacteroid
141 morphotypes according to the Aeschynomene host: E-morphotype in A. afraspera and the S-
142 morphotype in A. indica (Fig. 1A; Bonaldi et al., 2011). As this difference in shape could imply
143 different properties, we analyzed in each bacteroid type, isolated from mature nodules at 14 days post
144 inoculation (dpi), several characteristics that have been shown to change during bacteroid
145 metamorphosis in Medicago, i.e. the bacterial DNA content, the membrane permeability and the
146 viability. As revealed by flow cytometry analysis (Fig. 1B), the DNA content of E- and S- bacteroids
147 peaked respectively at around 7C and 16C in comparison to the 1C/2C DNA content of free-living
148 bacteria. These ploidy levels are less than described for S. meliloti bacteroids (24C) (Mergaert et al.,
149 2006) although other studies have shown lower ploidy levels also in Medicago (Sinharoy et al., 2013;
150 Berrabah et al., 2014).
151 Bacteroid membrane integrity was estimated using propidium iodide (PI), a DNA stain that is
152 excluded from living cells but penetrates in cells displaying membrane integrity damage (Mergaert et
153 al., 2006). Fluorescence images of the PI-stained bacteroids showed that the membrane permeability
154 of E- and S-morphotype bacteroids was more prominent than the one of the free-living bacteria (Fig.
155 1A). Indeed, 88% of S-bacteroids and 95% of E-bacteroids were found to be permeable to PI against
156 7% for free-living bacteria. For comparison, PI stained about 50% of S. meliloti bacteroids (Mergaert
157 et al., 2006). The loss of viability measured for S-morphotype bacteroids from A. indica nodules
158 (98%) was also comparable to the one previously estimated for S. meliloti bacteroids (99%) (Mergaert
159 et al., 2006). In contrast, intracellular cells of A. afraspera appeared more viable as 22 % formed
160 colonies on agar plates. However, this higher value could result from the fact that A. afraspera nodules
161 displayed the unusual presence of two infected tissues, a large central tissue in which the bacteria
162 elongated and a superficial tissue in which the shape of the bacteria remained unmodified (Bonaldi et
163 al., 2011). The intracellular cells extracted from nodules consisted therefore of a mixture of
164 differentiated and non-differentiated bacteria suggesting that the loss of viability of E-morphotype
165 bacteroids should be far more important. Taken together, we observed that the E- and S-morphotype
166 bacteroids from Aeschynomene nodules share the same features as the S. meliloti bacteroids in
167 Medicago and therefore, they can be considered as terminally differentiated.
168
169 Distribution of S- and E-Morphotype Bacteroids Among Aeschynomene Species
170 To obtain more insight into the distribution of the E- and S- bacteroid morphotypes among the
171 Aeschynomene species, we analyzed by confocal microscopy the shape of intracellular bacteria from
172 mature nodules of various Aeschynomene species elicited by the ORS285 strain tagged with GFP.
173 Unlike the other photosynthetic Bradyrhizobium strains, such as ORS278 and BTAi1, this bacterium
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174 does contain the canonical nodABC genes and displays a broader host range due to its ability to use a
175 Nod factor-dependent and a Nod factor-independent symbiotic process according the host plant
176 (Bonaldi et al., 2011). We observed in all tested species using a Nod factor-independent process (A.
177 indica, A. evenia ssp. serrulata, A. evenia. ssp. evenia, A. virginica, A. scabra, and A. sensitiva) that
178 the bacteroids displayed an S-morphotype whereas in the three Nod factor-dependent species tested (A.
179 afraspera, A. nilotica, A. aspera) the bacteroids displayed an E-morphotype (Fig. 2 and Supplemental
180 Fig S1B).
181 We also analyzed the bacteroid shape in another group of Aeschynomene species not nodulated by the
182 ORS285 strain but by non-photosynthetic Bradyrhizobium strains containing nod genes such as
183 ORS301, ORS302 and ORS305 (Molouba et al., 1999). We also observed in these species
184 (A. americana, A. pfundii, A. schimperi) that the bacteroids displayed an E-morphotype. Altogether
185 these data indicate that S-morphotype bacteroids are specific for the Aeschynomene species using a
186 Nod factor-independent symbiotic process, while the E-morphotype is specific to the Aeschynomene
187 using a Nod factor-dependent one.
188
189 Aeschynomene Species Contain a New Class of Nodule-Specific Cysteine-Rich Peptides-
190 Encoding Genes
191 Our results indicate that in Aeschynomene nodules, the endosymbiotic bacteria undergo terminal
192 differentiation similarly as described in nodules of IRLC species. This raises the question if NCR
193 peptides are also recruited in Aeschynomene to govern this differentiation step. To check this
194 possibility, we analyzed 4 expressed sequence tag (EST) libraries previously constructed in our
195 laboratory and corresponding to non-inoculated roots and mature nodules from A. afraspera and A.
196 indica plants inoculated with the ORS285 strain. Although the total number of cDNAs sequenced for
197 each library was relatively small, around 9500 ESTs/library (Supplemental Table S1), we postulated
198 that if NCR peptides were also involved in bacteroid differentiation in Aeschynomene, they should be
199 specifically and highly expressed in the nodules and hence easily detectable. We first performed a
200 BLAST search on the EST libraries using several M. truncatula NCR genes as a query but no
201 conclusive results were obtained. Considering that NCR genes are rapidly evolving and are highly
202 diverse, even within M. truncatula (Alunni et al., 2007; Branca et al., 2011), we re-analyzed the
203 available EST databases using the following parameters determined from typical features of the NCR
204 family: i) candidate genes should encode small proteins (less than 100 amino acids) containing a
205 signal peptide, ii) the gene expression should be nodule specific with a significant read count in the
206 EST database (we arbitrarily fixed a lower limit of 5 reads) and iii) the candidate genes should encode
207 peptides rich in Cys residues (at least 4). This second in silico analysis identified 15 genes encoding
208 putative NCR-like peptides from the A. afraspera nodule library and 18 from the library of A. indica
209 nodules (Fig. 3).
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210 These genes were named Aa- or AiNCRs and numbered according their rank in read counts
211 (Supplemental Table S2). The deduced mature peptides (i.e. without their signal peptide) are between
212 37 and 85 residues long and contain 4 to 10 Cys (Fig. 3A). The alignment of these NCR-like peptides
213 highlighted, for 31 out of the 33 sequences, a conserved pattern of 6 Cys within the sequence of the
214 mature peptides (Fig. 3B). This pattern is very close to the one previously described for a group of
215 Medicago NCRs but distinct by the spacing between the first three Cys. This led us to propose a
216 specific Aeschynomene NCR-like motif. Interestingly, 11 of these NCR-like peptides contained 2
217 extra-Cys, giving rise to a typical defensin signature that was named motif 2 (Fig. 3B).
218 By analogy with M. truncatula, for which the NCR family is composed of about 600 genes that exhibit
219 distinct temporal and spatial expression patterns in nodules (Mergaert et al., 2006; Alunni et al., 2007;
220 Young et al., 2011; Guefrachi et al., 2014), the involvement of a larger number of NCR genes is also
221 expected in Aeschynomene. We therefore considered the possibility that our criteria were too selective
222 leading to a restricted number of candidate genes identified. By performing BLAST searches against
223 the same EST libraries but using this time the first candidate genes that emerged, we identified a total
224 of 38 and 44 NCR-like genes in A. afraspera and A. indica, respectively (Supplemental Table S2). All
225 these additional genes were found to be specifically expressed in the nodules and a global alignment
226 show that all of them except 3 contained the Aeschynomene NCR-motif 1 or 2.
227 Furthermore, a BLAST search against transcriptome data obtained from A. evenia ssp. serrulata
228 mature nodules made by the 454 technology which allows the detection of a larger number of
229 transcripts, revealed an even higher number of NCR-like genes of more than 80 (Supplemental Table
230 S2). Altogether, these data suggest that the NCR-like genes constitute also in Aeschynomene an
231 important family that could count several tens or even hundreds of members.
232
233 NCR-Like Genes of Aeschynomene sp. are Expressed Before or Concomitantly with
234 Bacteroid Differentiation
235 In order to analyze the temporal expression of the Aa- and AiNCR genes, we performed an RT-qPCR
236 analysis of 4 of them for each Aeschynomene species. Root tissues of A. afraspera and A. indica were
237 sampled at T0 and at different time points after inoculation with Bradyrhizobium strain ORS285 (6h, 1,
238 2, 3, 4, 5, 6, 7 and 14 days). As a symbiotic marker, we also monitored Leghemoglobin (LegHb)
239 mRNA accumulation. As shown in Figures 4A and 4B, mRNA corresponding to LegHb started to
240 accumulate at 5 dpi for both species, i. e. when the bacteroid differentiation process is completed and a
241 nitrogenase activity is detectable (Bonaldi et al., 2011). Interestingly, while the NCR-like gene
242 transcripts were not detected in control roots or during the early time points of nodule formation, they
243 were all strongly expressed at 5dpi and some of them even started to accumulate 1 or 2 days before
244 (Fig. 4A and B), which coincided with the beginning of bacteroid morphogenesis (Bonaldi et al.,
245 2011).
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246 To complete this expression analysis, we also checked the expression by RT-qPCR, in both roots and
247 nodules, of 3 candidates NCR genes identified in A. evenia by BLAST. As expected, the 3 genes
248 appeared specifically and highly expressed in A. evenia nodules (Supplemental Fig. S2).
249
250 NCR-Like Genes are Expressed Specifically in Infected Cells of Aeschynomene sp.
251 Nodules
252 In Medicago, the NCRs are specifically synthetized in the infected plant cells and are targeted to the
253 bacteria (Van de Velde et al., 2010). To check if the expression of the identified Aa- and AiNCR genes
254 was also restricted to the infected cells, we performed an in situ hybridization on 14-days old nodules
255 of A. afraspera and A. indica, infected with the ORS285 strain. We used as probes AaNCR01 and
256 AiNCR01, which were found to be the most highly expressed NCR genes in each species. In addition,
257 the LegHb gene, known to be expressed in infected nodule cells, was included as a positive control for
258 the hybridization experiment. As shown in Fig. 5, a strong signal was obtained using the LegHb
259 antisense DNA as a probe. The signal was restricted to the infected cells (Fig. 5B and E), consistent
260 with the known role of this protein in symbiosis. Both A. afraspera and A. indica NCR antisense
261 probes gave also a signal limited to the infected cells (Fig. 5C and F) similarly as observed for LegHb.
262 Control hybridizations without probe or with a sense LegHb probe or sense NCR-like probes (Fig. 5A
263 and D) showed a complete absence of signal.
264
265 NCR-Like Peptides Target Bacteroids in Aeschynomene Nodules
266 To test the possibility that Aa- and AiNCR mature peptides are targeted to the endosymbiotic bacteria,
267 we set up a proteome approach. Total protein extracts were prepared from nodules and from purified
268 bacteroids from both A. afraspera and A. indica. In addition, a control extract was prepared from free-
269 living ORS285 bacteria. Proteins were subjected to Tricine-PAGE analysis. Slight differences in the
270 patterns of low molecular weight proteins (between 4 to 9 kDa), where we expected the Aa- or AiNCR
271 peptides, could be observed between nodule or bacteroid extracts and extracts from free-living bacteria
272 (Supplemental Fig. S3). We identified the corresponding proteins by mass spectrometry. From the
273 bacteroid extracts isolated from A. afraspera and A. indica nodules, 51 and 42 proteins were identified
274 respectively (Supplemental Table S3). Among them, in both nodule contexts, 4 corresponded to plant
275 proteins, of which three were identified as NCR peptides (Table 1). The additional plant protein
276 identified in A. indica bacteroids was a putative monosaccharide-proton symporter corresponding
277 probably to a symbiosome located transporter whereas there was an extensin-like protein of unknown
278 function in A. afraspera bacteroids. Among the 40 proteins identified in free-living bacterial cells, all
279 corresponded to bacterial proteins and none displayed the characteristics of cysteine-rich peptides
280 (Supplemental Table S3).
281
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