the t3ss of cupriavidus taiwanensis strain lmg19424 ...ut in the absence of reduced nitrogen, most...

Symbiotic role of C. taiwanensis T3SS / AEM 2012

- 1 -

1 The T3SS of Cupriavidus taiwanensis strain LMG19424 2 compromizes symbiosis with Leucaena leucocephala 3

4 Maged M. Saad1,2, Michèle Crèvecoeur1, Catherine Masson-Boivin2 and Xavier 5 Perret*1 6 1 University of Geneva, Sciences III, Department of Botany and Plant Biology, 30 quai 7

Ernest-Ansermet, CH-1211 Geneva 4, Switzerland 8 2 Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR CNRS-INRA 9

2594/441, BP 52627, 31326 Castanet-Tolosan Cedex, France 10 11 Running Title: T3SS and host-range of C. taiwanensis 12 Keywords: nitrogen fixation, nodules, NGR234, host-range. 13 Section: genetics and molecular biology. 14 15 16 * To whom correspondence should be addressed : 17

Xavier Perret 18 University of Geneva, Sciences III 19 Department of Botany and Plant Biology, Microbiology Unit, 20 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. 21 Tel. +41 – 22 – 379 – 3117 22 Fax +41 – 22 – 379 – 3199 23 Email [email protected] 24 25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01691-12 AEM Accepts, published online ahead of print on 3 August 2012

on June 6, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 2 -

ABSTRACT 26 Cupriavidus taiwanensis forms proficient symbioses with few Mimosa species. Inactivation of 27 T3SS had no effect on M. pudica, but allowed C. taiwanensis to establish chronic infections 28 and fix nitrogen in Leucaena leucocephala. Unlike in other rhizobia, glutamate rather than 29 plant flavonoids mediates transcriptional activation of this atypical T3SS. 30

31


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 3 -

In the absence of reduced nitrogen, most legumes species form nitrogen-fixing 32 associations with soil bacteria collectively known as rhizobia. Symbioses between rhizobia 33 and legumes come in many forms and shapes (11), but always culminate with the formation 34 on roots (or stems) of specialized organs called nodules. To limit risks of detrimental 35 infections as well as maximize nitrogen fixation, plants restrict the intracellular colonization by 36 rhizobia to specialized nodule cells. Within these nodule cells, rhizobia differentiate into 37 nitrogen-fixing bacteroids that exchange ammonia for carbohydrates derived from 38 photosynthates (13). Colonization of plant tissues by rhizobia involves the exchange between 39 both symbionts of molecular codes such as flavonoids, nodulation-factors, and surface 40 polysaccharides (6, 14). Rhizobia are phylogeneticaly disparate bacteria distributed in many 41 genera of α- and β-proteobacteria, referred to as α- and β-rhizobia. In several distantly 42 related α-rhizobia such as Sinorhizobium fredii strain NGR234 (16) or Bradyrhizobium 43 japonicum strain 110 (8), type three protein secretion systems (T3SS) were shown to 44 promote or impair symbiosis with host plants (4), including at the cultivar level (18). Whether 45 β-rhizobia also use T3SS to modulate their host range had not been investigated so far. 46

The T3SS of Cupriavidus taiwanensis is an atypical rhizobial secretion system. 47 The β-rhizobium Cupriavidus taiwanensis LMG19424 is a specific symbiont of Mimosa 48 species, including M. pudica. Genome sequencing revealed that LMG19424 carries a T3SS 49 cluster of unknown function on its chromosome 2 (1). Genes required for type III secretion 50 and cellular translocation (sct) are clustered into two groups of genes divergently transcribed, 51 the largest of which covers 12.2 kb and codes for sctVSCDJKLNTU. Interestingly this genetic 52 organization differs markedly from that found in α-rhizobia, but resembles that of the human 53 opportunist Burkholderia cenocepacia (1). In most rhizobia transcription of both nodulation 54 (nod, nol, noe) and T3SS (sct) genes is controlled via flavonoid-dependent regulatory 55 cascades (4, 7). To study the expression of C. taiwanensis T3SS genes, the promoter of 56 sctV (PsctV) was amplified and fused to lacZ of pCZ388 (3) yielding pCZ-PsctV. Once 57 mobilized into CBM832, a streptomycin-resistant derivative of LMG19424 (10), the activity of 58


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 4 -

pCZ-PsctV was monitored in free-living cells grown in quarter-strength minimal medium 59 (MM/4-S) supplemented with 10 mM succinate as carbon source and vitamins (10). In these 60 growth conditions, the PsctV-lacZ fusion was not induced by luteolin or apigenin, two 61 inducers of LMG19424 nodulation genes such as nodB (Table 1). In contrast, the addition of 62 glutamate triggered the activity of PsctV, but not that of a nodB-lacZ fusion (pCBM01). 63 Although presence of glutamate was reported to induce the expression of T3SS functions in 64 cells of Pseudomonas aeruginosa and Ralstonia solanacearum grown in vitro in minimal 65 media (e.g. see 3), signals responsible for the in vivo activation of sct loci in both pathogens 66 remain unknown. Thus, our data confirmed that pCZ-PsctV was functional and, unlike what 67 was found for α-rhizobia, regulation of T3SS genes in C. taiwanensis was not mediated by 68 flavonoids capable of inducing nod gene expression. 69 The T3SS of C. taiwanensis has no effect on M. pudica. To examine the role of C. 70 taiwanensis T3SS in symbiosis, a polar mutation was introduced in sctN (RALTA_B1253), 71 which encodes an ATPase required for T3SS-dependent secretion of proteins (4). A 72 fragment internal to sctN was amplified by PCR using CBM832 genomic DNA and primers 73 HindIII-F_SctN (5’-ccaagcttgatccggtggacaacgaac-3’) and BamHI-R_SctN (5’-74 cgggatcccgatatggccgtcgaggatg-3’). The 647 bp amplicon was cloned into suicide vector 75 pVO155 (12) digested with BamHI and HindIII, yielding pVOsctN. Once mobilized into 76 CBM832 by tri-parental mating, single-reciprocal recombination of pVOsctN with 77 chromosome 2 was selected using resistance to neomycin. Southern hybridization and PCR 78 amplifications confirmed the genotype of the sctN-mutant strain CBM312, in which pVO155 79 separates sctN into two truncated fragments of 1,035 and 348 bp. 80

The phenotype of mutant strain CBM312 was assessed first on Mimosa pudica, the 81 primary host of C. taiwanensis (2). Plants were grown in Magenta jars containing vermiculite, 82 watered using nitrogen-free B&D solution and inoculated with 2 x 108 bacteria per 83 germinated seedlings (5). As shown in Figure 1, both CBM312 and CBM832 elicited root 84 nodules in which cells of the central nitrogen-fixation zone were massively infected with 85


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 5 -

bacteria. Bacteria re-isolated from nodules were found to be resistant to neomycin and to 86 carry a copy of pVO155 inserted in sctN. Electron micrographs of nodule sections also 87 confirmed that bacteroids of CBM312 and CBM832 appeared similar in size and shape (Fig. 88 1B and 1D). At 45 days post-inoculation the average number of nodules, nodule fresh-89 weight, and shoot dry-weight of plants inoculated with either CBM312 or CBM832 were 90 similar, indicating that a functional T3SS was not required for a proficient symbiosis on M. 91 pudica (Table 2). Additional nodulation tests confirmed that both CBM832 and CBM312 92 failed to form nodules on roots of Vigna unguiculata and Pachyrhizus tuberosus, and induced 93 non-fixing pseudonodule-like structures on Tephrosia vogelii and Crotalaria juncea roots. 94

A functional T3SS restricts the host range of C. taiwanensis. Native to southern 95 Mexico and northern Central America, L. leucocephala is a species of small trees that 96 belongs to the same Mimoseae tribe as M. pudica. L. leucocephala (Lam.) de Wit forms 97 nitrogen-fixing associations with various rhizobial species (17), including the promiscuous 98 Sinorhizobium fredii strain NGR234 (15), but not with a closely related S. fredii strain 99 USDA257 that lacks a functional copy of the nodSU locus (9). NodS and NodU that add 100 respectively methyl and carbamoyl groups onto Nod-factors are present in both strains 101 NGR234 and C. taiwanensis LMG19424 (1). In addition, a number of α-rhizobia strains that 102 were isolated from nodules of Acacia but that nodulate L. leucocephala, were shown to 103 synthesize Nod factors that were structurally similar to those made by C. taiwanensis (1). On 104 L. leucocephala, the parent strain CBM832 formed small nodules that were poorly infected, 105 however (see Fig. 2A). Inter- as well as intra-cellular structures resembling infection-threads 106 or -pockets were observed (see Fig. S1 in supplemental material), but overall few nodule 107 cells contained intracellular CBM832 symbiosomes and these appeared often to be in the 108 process of being degraded (Fig. 2B). Nodules also lacked leghemoglobin, failed to fix 109 nitrogen, and ultimately plants were starved for nitrogen (Table 1). In contrast, nodules 110 formed by mutant strain CBM312 fixed nitrogen, and had well-defined meristematic zones 111 (Fig. 2C) as well as mature nodule cells with densily-packed bacteroids (Fig. 2D). This 112


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 6 -

indicated that a functional T3SS impaired the persistent colonization of nodule cells rather 113 than the processes of nodulation and infection on plant tissues per se. When compared to 114 NGR234, CBM312 seemed a less efficient symbiont as the shoot dry-weight of plants was 115 significantly lower (Table 1). Interestingly, the T3SS of NGR234 was reported to have no 116 measurable effect on L. leucocephala (4, 16). 117

Thus, a functional T3SS prevents symbiosis with L. leucocephala and contributes to 118 restrict the host range of C. taiwanensis. Rhizobia evolved in many unrelated genera most 119 probably by acquiring sets of symbiotic genes via lateral transfer followed by a 120 reprogramming of the recipient genome to express and optimize these symbiotic traits (11). 121 In this respect, strain LMG19424 was shown to harbour the most compact symbiotic island 122 described so far (1), suggesting that it evolved recently from a non-symbiotic ancestor who’s 123 closely related to a bacterium capable of infecting humans (2). A genetic organization similar 124 to T3SS of pathogenic bacteria, a transcriptional activation not mediated by flavonoids, and 125 an activity that compromises colonization of nodule cells by bacteroids, suggest that 126 integration of C. taiwanensis T3SS in the symbiotic lifestyle was not complete in a micro-127 symbiont that is still in the making. 128 129 ACKNOWLEDGEMENTS 130

We would like to thank Natalia Giot, Carine Gris, and Anne Utz for their help in many 131 aspects of this work. MS was supported by a post-doctoral fellowship from INRA SPE. XP 132 acknowledges financial support by the University of Geneva and the Swiss National Science 133 Foundation (grant n°31003A-116591). Work in CMB laboratory is part of the "Laboratoire 134 d'Excellence” (LABEX) entitled TULIP (ANR-10-LABX-41) and was supported by a grant 135 from SPE INRA and ANR-08-BLAN-0295-01. 136 137


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 7 -

REFERENCES 138 1. Amadou, C., G. Pascal, S. Mangenot, M. Glew, C. Bontemps, D. Capela, S. Carrere, S. 139

Cruveiller, C. Dossat, A. Lajus, M. Marchetti, V. Poinsot, Z. Rouy, B. Servin, M. Saad, C. 140 Schenowitz, V. Barbe, J. Batut, C. Medigue, and C. Masson-Boivin. 2008. Genome sequence 141 of the β-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Res. 142 18:1472-1483. 143

2. Chen, W. M., S. Laevens, T. M. Lee, T. Coenye, P. De Vos, M. Mergeay, and P. Vandamme. 144 2001. Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of 145 a cystic fibrosis patient. Int. J. Syst. Evol. Microbiol. 51:1729-1735. 146

3. Cunnac, S., C. Boucher, and S. Genin. 2004. Characterization of the cis-acting regulatory 147 element controlling HrpB-mediated activation of the type III secretion system and effector genes in 148 Ralstonia solanacearum. J. Bacteriol. 186:2309-2318. 149

4. Deakin, W. J., and W. J. Broughton. 2009. Symbiotic use of pathogenic strategies: rhizobial 150 protein secretion systems. Nat. Rev. Microbiol. 7:312-320. 151

5. Fumeaux, C., N. Bakkou, J. Kopcinska, W. Golinowski, D. J. Westenberg, P. Muller, and X. 152 Perret. 2011. Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain 153 NGR234 using a transposon with a NifA-dependent read-out promoter. Microbiol. 157:2745-2758. 154

6. Gibson, K. E., H. Kobayashi, and G. C. Walker. 2008. Molecular determinants of a symbiotic 155 chronic infection. Annu. Rev. Genet. 42:413-441. 156

7. Kobayashi, H., Y. Naciri-Graven, W. J. Broughton, and X. Perret. 2004. Flavonoids induce 157 temporal shifts in gene-expression of nod-box controlled loci in Rhizobium sp. NGR234. Mol. 158 Microbiol. 51:335-347. 159

8. Krause, A., A. Doerfel, and M. Göttfert. 2002. Mutational and transcriptional analysis of the type 160 III secretion system of Bradyrhizobium japonicum. Mol. Plant-Microbe Interact. 15:1228-1235. 161

9. Krishnan, H. B., A. Lewin, R. Fellay, W. J. Broughton, and S. G. Pueppke. 1992. Differential 162 expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium 163 sp. strain NGR234 to nodulate Leucaena spp. Mol. Microbiol. 6:3321-3330. 164

10. Marchetti, M., D. Capela, M. Glew, S. Cruveiller, B. Chane-Woon-Ming, C. Gris, T. Timmers, V. 165 Poinsot, L. B. Gilbert, P. Heeb, C. Medigue, J. Batut, and C. Masson-Boivin. 2010. 166 Experimental evolution of a plant pathogen into a legume symbiont. PLoS Biol. 8:e1000280. 167

11. Masson-Boivin, C., E. Giraud, X. Perret, and J. Batut. 2009. Establishing nitrogen-fixing 168 symbiosis with legumes: how many rhizobium recipes? Trends Microbiol. 17:458-466. 169

12. Oke, V., and S. R. Long. 1999. Bacterial genes induced within the nodule during the Rhizobium-170 legume symbiosis. Mol. Microbiol. 32:837-849. 171

13. Oldroyd, G. E., J. D. Murray, P. S. Poole, and J. A. Downie. 2011. The rule of engagement in the 172 legume-rhizobial symbiosis. Annu. Rev. Genet. 45:119-144. 173

14. Perret, X., C. Staehelin, and W. J. Broughton. 2000. Molecular basis of symbiotic promiscuity. 174 Microbiol. Mol. Biol. Rev. 64:180-201. 175


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 8 -

15. Pueppke, S. G., and W. J. Broughton. 1999. Rhizobium sp. strain NGR234 and R. fredii 176 USDA257 share exceptionally broad, nested host ranges. Mol. Plant-Microbe Interact. 12:293-318. 177

16. Viprey, V., A. Del Greco, W. Golinowski, W. J. Broughton, and X. Perret. 1998. Symbiotic 178 implications of type III protein secretion machinery in Rhizobium. Mol. Microbiol. 28:1381-1389. 179

17. Wang, E. T., J. Martínez-Romero, and E. Martínez-Romero. 1999. Genetic diversity of rhizobia 180 from Leucaena leucocephala nodules in Mexican soils. Mol. Ecol. 8:711-724. 181

18. Yang, S., F. Tang, M. Gao, H. B. Krishnan, and H. Zhu. 2010. R gene-controlled host specificity 182 in the legume-rhizobia symbiosis. Proc. Natl. Acad. Sci. USA 107:18735-18740. 183

184 185


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 9 -

Table 1. Glutamate rather than flavonoids activates T3SS genes of C. taiwanensis. 186 The promoter region of sctV was amplified using primers PsctV_F (5’-ccaagcttatcaggctccatatgcgg-3’) 187 and PsctV_R (5’-aactgcagatcacggcaaacagcagca-3’), cloned into pGEM-Teasy (Promega) as one 188 HindIII-PstI fragment and further subcloned into pCZ388 using the same restriction enzymes. The 189 resulting PsctV-lacZ transcriptional fusion (pCZ-PsctV) was introduced into CBM832 by conjugation. 190 β-galactosidase activities of pCZ-PsctV, a nodB-lacZ fusion (pCBM01) (10), and empty vector pCZ388 191 (3) were measured in transconjugants of CBM832 grown for 24 h in MM/4-S supplemented with 192 vitamins and 10 mM succinate using apigenin (5 µM), luteolin (15 µM), or glutamate (90 mM) as 193 inducers. Values are reported as Miller’s units and represent the means of at least 3 independent 194 experiments. Standard deviations are shown in brackets. 195 β-galactosidase activities of constructs in MM/4-S medium containing

Construct no-inducer apigenin luteolin glutamate

pCZ388 25.8 (±7.3) 18.5 (±8.8) 20.2 (±10.1) 28.9 (±3.9)

pCBM01 89.6 (±8.8) 2,689 (±322) 2,287 (±125) 128.0 (±34.1)

pCZ-PsctV 35.4 (±3.9) 17.3 (±9.7) 32.4 (±4.1) 591.4 (±53.9)

196 197


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 10 -

Table 2. Symbiotic properties of CBM312, CBM832 and NGR234. 198 Symbiotic properties of inoculated strains are reported as the mean nodule number (mNN), nodule 199 fresh weight (mNFW), and shoot dry weight (mSDW) per inoculated plant, 45 or 50 days post 200 inoculation (dpi). Results are the means of at least 10 plants per inoculant, with the standard 201 deviations shown in brackets. mSDW of non-inoculated control plants was of 79.6 (±17.5), and 10.8 202 (±1.7) mg for L. leucocephala and M. pudica, respectively. 203

Host plant Inoculant mNN mNFW (mg) mSDW (mg)

M. pudica

(45 dpi)

CBM312 48.5 (±15.6) 58.7 (±12.9) 157.5 (±83.8)

CBM832 49.0 (±16.1) 62.4 (±19.3) 151.8 (±40.1)

NGR234 0.0 0.0 14.7 (±4.8)

L. leucocephala

(50 dpi)

CBM312 23.3 (±6.1) 226.7 (±71.8) ‡ 209.2 (±53.7) ‡

CBM832 21.9 (±6.1) 77.1 (±21.0) 95.9 (±20.5)

NGR234 19.5 (±4.4) 170.1 (±39.1) § 283.0 (±68.8) §

204 ‡ Values obtained with CBM312 that are significantly different from that of CBM832 at the level α=5%. 205 § Values obtained with NGR234 that are significantly different from that of CBM312 at the level α=5%. 206 207 on June 6, 2020 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/


- 11 -

FIGURES and their LEGEND 208 209 210 211 212 213 FIG. 1. Sections of nodules formed by CBM832 (A, B) or CBM312 (C, D) on roots of M. 214 pudica 35 days post-inoculation. A and C, cross-sections of nodules seen at low 215 magnification (scale bars, 500 µm) using light microscopy. B and D, electron micrographs of 216 nodule cells containing bacteroids (scale bars, 2 µm). 217

218


.asm.org/

Dow

nloaded from

http://aem.asm.org/


- 12 -

219 220 221 222 223 224 225 226 227 228 229 230 231 FIG. 2. Sections of nodules formed by CBM832 (A, B) or CBM312 (C, D) on roots of L. 232 leucocaphala 35 days post-inoculation. A and C, light micrographs of nodule sections at a 233 low magnification (scale bars, 500 µm). B and D, electron micrographs of nodule cells 234 containing intracellular bacteria of CBM832 (B) or nitrogen-fixing bacteroids of CBM312 235 (scale bars, 2 µm). 236

237


.asm.org/

Dow

nloaded from

http://aem.asm.org/


.asm.org/

Dow

nloaded from

http://aem.asm.org/

the t3ss of cupriavidus taiwanensis strain lmg19424 ...ut in the absence of reduced nitrogen, most...

Documents