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The identification of novel loci required for appropriate nodule development in Medicago truncatula.

Domonkos A, Horvath B, Marsh JF, Halasz G, Ayaydin F, Oldroyd GE, Kalo P - BMC Plant Biol. (2013)

Bottom Line: Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci.Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő 2100, Hungary. kalo@abc.hu.

ABSTRACT

Background: The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.

Results: Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

Conclusions: The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

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The degree of rhizobial infection in Fix- mutants. Nodule sections of wild-type (A) and Fix- mutants (B-I). Nodules were harvested 3 weeks post inoculation with S. meliloti 1021 expressing the lacZ gene. 70 μm thick nodule sections were stained with X-gal to detect β-galactosidase activity. Nodules of (B) ipd3-1, (C)dnf5-2, (D) 7Y, (E) 5L, (F) 11S, (G)dnf8, (H) 13U and (I) dnf7-2 mutants show various degrees of bacterial colonization. Roman letters on wild-type nodules (A) indicate the zones of a mature nodule: I., meristem; II., infection zone; II-III., interzone; III., nitrogen-fixation zone. The arrow shows an infection thread in panel G. Bars represent 200 μm.
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Figure 3: The degree of rhizobial infection in Fix- mutants. Nodule sections of wild-type (A) and Fix- mutants (B-I). Nodules were harvested 3 weeks post inoculation with S. meliloti 1021 expressing the lacZ gene. 70 μm thick nodule sections were stained with X-gal to detect β-galactosidase activity. Nodules of (B) ipd3-1, (C)dnf5-2, (D) 7Y, (E) 5L, (F) 11S, (G)dnf8, (H) 13U and (I) dnf7-2 mutants show various degrees of bacterial colonization. Roman letters on wild-type nodules (A) indicate the zones of a mature nodule: I., meristem; II., infection zone; II-III., interzone; III., nitrogen-fixation zone. The arrow shows an infection thread in panel G. Bars represent 200 μm.

Mentions: In order to assess the stage at which the defects occurred in the Fix- mutants, we inoculated with S. meliloti 1021 (pXLGD4) which constitutively expresses the LacZ reporter gene (hemA::lacZ; [31]). The Fix- mutants developed exclusively white spherical or slightly cylindrical nodules except dnf8 on which an occasional pale pink nodule could be observed. To visualize the presence of bacteria in the nodules we stained longitudinal sections of 21-day-old nodules following assays for β-galactosidase activity. The extent of bacterial colonization in the nodule zones was examined by light microscopy (Figure 3A-I). Wild type nodules showed typical zonation [5] (Figure 3A) and no such zonation was observed in nodules of ipd3-1 and dnf5-2 (Figure 3B and C). The majority of the nodules formed on ipd3-1 roots were spherical with abnormal nodule apices (Figure 3B), but a small number of nodules developed into elongated cylindrical structures [30]. Neither class of ipd3-1 nodules contained cells with released bacteria, indicating the essential function of IPD3 for bacterial release. In contrast to ipd3-1, the dnf5-2 and dnf7-2 nodules had cells containing bacteria (Figure 3C and I), but no characteristic zonation of the indeterminate nodules was observed in dnf5-2 nodules (Figure 3C) and the nitrogen-fixation zone was devoid of rhizobia except a few sporadic infection threads (Figure 3I) in dnf7-2 nodules.


The identification of novel loci required for appropriate nodule development in Medicago truncatula.

Domonkos A, Horvath B, Marsh JF, Halasz G, Ayaydin F, Oldroyd GE, Kalo P - BMC Plant Biol. (2013)

The degree of rhizobial infection in Fix- mutants. Nodule sections of wild-type (A) and Fix- mutants (B-I). Nodules were harvested 3 weeks post inoculation with S. meliloti 1021 expressing the lacZ gene. 70 μm thick nodule sections were stained with X-gal to detect β-galactosidase activity. Nodules of (B) ipd3-1, (C)dnf5-2, (D) 7Y, (E) 5L, (F) 11S, (G)dnf8, (H) 13U and (I) dnf7-2 mutants show various degrees of bacterial colonization. Roman letters on wild-type nodules (A) indicate the zones of a mature nodule: I., meristem; II., infection zone; II-III., interzone; III., nitrogen-fixation zone. The arrow shows an infection thread in panel G. Bars represent 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852326&req=5

Figure 3: The degree of rhizobial infection in Fix- mutants. Nodule sections of wild-type (A) and Fix- mutants (B-I). Nodules were harvested 3 weeks post inoculation with S. meliloti 1021 expressing the lacZ gene. 70 μm thick nodule sections were stained with X-gal to detect β-galactosidase activity. Nodules of (B) ipd3-1, (C)dnf5-2, (D) 7Y, (E) 5L, (F) 11S, (G)dnf8, (H) 13U and (I) dnf7-2 mutants show various degrees of bacterial colonization. Roman letters on wild-type nodules (A) indicate the zones of a mature nodule: I., meristem; II., infection zone; II-III., interzone; III., nitrogen-fixation zone. The arrow shows an infection thread in panel G. Bars represent 200 μm.
Mentions: In order to assess the stage at which the defects occurred in the Fix- mutants, we inoculated with S. meliloti 1021 (pXLGD4) which constitutively expresses the LacZ reporter gene (hemA::lacZ; [31]). The Fix- mutants developed exclusively white spherical or slightly cylindrical nodules except dnf8 on which an occasional pale pink nodule could be observed. To visualize the presence of bacteria in the nodules we stained longitudinal sections of 21-day-old nodules following assays for β-galactosidase activity. The extent of bacterial colonization in the nodule zones was examined by light microscopy (Figure 3A-I). Wild type nodules showed typical zonation [5] (Figure 3A) and no such zonation was observed in nodules of ipd3-1 and dnf5-2 (Figure 3B and C). The majority of the nodules formed on ipd3-1 roots were spherical with abnormal nodule apices (Figure 3B), but a small number of nodules developed into elongated cylindrical structures [30]. Neither class of ipd3-1 nodules contained cells with released bacteria, indicating the essential function of IPD3 for bacterial release. In contrast to ipd3-1, the dnf5-2 and dnf7-2 nodules had cells containing bacteria (Figure 3C and I), but no characteristic zonation of the indeterminate nodules was observed in dnf5-2 nodules (Figure 3C) and the nitrogen-fixation zone was devoid of rhizobia except a few sporadic infection threads (Figure 3I) in dnf7-2 nodules.

Bottom Line: Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci.Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő 2100, Hungary. kalo@abc.hu.

ABSTRACT

Background: The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.

Results: Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.

Conclusions: The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

Show MeSH
Related in: MedlinePlus