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. 2013 Feb;9(2):e1003204.
doi: 10.1371/journal.ppat.1003204. Epub 2013 Feb 28.

Mutualistic Co-Evolution of Type III Effector Genes in Sinorhizobium Fredii and Bradyrhizobium Japonicum

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Free PMC article

Mutualistic Co-Evolution of Type III Effector Genes in Sinorhizobium Fredii and Bradyrhizobium Japonicum

Jeffrey A Kimbrel et al. PLoS Pathog. .
Free PMC article

Abstract

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Within-group genetic diversity for S. fredii, B. japonicum, is higher than the diversity within the P. syringae groups.
A rooted tree was constructed from the concatenated sequences of 103 genes present in all 17 strains and Geobacter sulfurreducens PCA and Desulfovibrio vulgaris used as outgroups. The scale bar indicates the number of amino acid substitutions per site. Phylogenetic divergence (PD) was measured for each group and compared to randomly assigned groups of strains. Reliable SNPs, based on pairwise comparisons to group-specific reference strains (*), were identified and calculated per kb (see Figure S1). The percent orthology was averaged from all within-group pairwise comparisons (see Figure S2). Each group included strains with finished (underlined) and draft genome sequences.
Figure 2
Figure 2. PtoDC3000 delivers T3Es of rhizobia in a T3SS-dependent manner.
(A) Leaves of Arabidopsis Col-0 (Rps2/Rps2) were infiltrated with PtoDC3000 (top row) and its T3SS-deficient mutant, ΔhrcC (bottom row) carrying no fusion to Δ79avrRpt2 or fusions to P. syringae T3E avrRpm1 or coding sequences from NGR234 candidate T3E genes, nopJ or nopB. (B) Members of the NopB T3E gene family all encode for functional T3Es. Leaves of Arabidopsis Col-0 (Rps2/Rps2) were infiltrated with PtoDC3000 carrying no fusion to Δ79avrRpt2 or fusions to P. syringae T3E avrRpm1 or nopB coding sequences from NGR234, USDA207, or USDA110. Leaves did not respond to infiltrations of ΔhrcC. In all experiments, leaves were scored for the HR ∼20 hpi and the percent of responding leaves are presented (at least 20 leaves infiltrated). Experiments were repeated at least three times.
Figure 3
Figure 3. Distribution of T3E families in rhizobia.
The T3E family names are listed across the top with strains of (A) S. fredii and (B) B. japonicum listed down the side. Boxes are color-coded as indicated in the key; white boxes = no detectable homolog. Conservation of T3Es is also color-coded (bars below each chart) as indicated.
Figure 4
Figure 4. T3E collections of S. fredii and B. japonicum are highly conserved in content.
(A) Representation of T3Es in categories as percentage of total number of T3E families in S. fredii, B. japonicum, as well as group I and legume pathovars of P. syringae. Unconfirmed T3Es were not included. (B) Fisher's exact test for all pairwise comparisons (connected by lines) of the representation of T3Es in the four categories depicted in panel (A). Boxed p-values are significant (Bonferonni adjusted α level = 0.0083). (C) Numbers of T3E genes and all genes binned as total, core, or accessory for each group of bacteria. Core genes are defined as those with orthologs present in all strains within each group. A Fisher's exact test was used to test for differences in distribution of core and accessory T3E genes relative to the distribution of all genes. All p-values are significant (Bonferonni adjusted α level = 0.0125).
Figure 5
Figure 5. T3E of S. fredii and B. japonicum have high levels of within-family amino acid identity.
(A) Balloon plots of within-family amino acid conservation for translated T3Es. The percent amino acid identity was calculated for all pairwise comparisons within each family (y-axis) and plotted according to the number of members within families (x-axis). The sizes of the balloons are scaled with the largest representing 162 pairwise comparisons (the smallest balloons were enlarged). Unconfirmed T3E and pseudogene sequences were not included in the comparisons. (B) Kolmogorov–Smirnov test for all pairwise comparisons (connected by lines) of the distributions depicted in panel (A). Boxed p-values are significant (Bonferonni adjusted α level = 0.0083). (C) An F test for linear hypothesis was used to test for differences in percent amino acid identity for translated T3Es and core genes within each group. All p-values are significant (Bonferonni adjusted α level = 0.0125).

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Grant support

This work was supported by the National Research Initiative Competitive Grants Program Grant no. 2008-35600-04691 and the Agriculture Research Foundation to JHC and National Science Foundation grant nos. 0816663 and 1150278 to JLS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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