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Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens

ABSTRACT A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbioses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C35) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid membrane resulting from the absence of free or lipidA-bound C35 hopanoids or the accumulation of the C30 hopanoid diploptene. Our results also show that C35 hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage.

a nuanced understanding of the biological functions of diverse hopanoids in modern niches.
The capacity for hopanoid biosynthesis is statistically enriched in the (meta-)genomes of bacteria associated with plants (9), and hopanoids have been found in high abundance in membranes of well-studied plant symbionts of the Bradyrhizobium (40% of total lipid extract [TLE]) and Frankia (87%) genera (10,11). Hopanoids promote membrane rigidity (12) and confer protection against numerous stresses, including acidic or alkaline pH, high temperature, high osmolarity, oxidative stress, detergents, and antibiotics (13)(14)(15)(16). They have varied structures, formed via methylation (2Me or 3Me), unsaturation, and/or attachment to a ribose-derived side chain (C 35 ) ( Fig. 1A; also see Fig. S1 in the supplemental material) (7). Whether there are functional distinctions under specific environmental conditions for diverse hopanoid types is unclear, yet some evidence suggests that they have nonoverlapping roles. For example, in Rhodopseudomonas palustris (17) and Burkholderia cenocepacia (14), C 35 hopanoids are critical for OM stability and for resistance to low pH, detergent (sodium dodecyl sulfate [SDS]), and polymyxin B, respectively. Though the absence of 2Me-hopanoids did not manifest a stress phenotype in previous tests of R. palustris, their biosynthesis is transcriptionally induced under stress (13). This suggests that 2Me-hopanoids might contribute to stress resistance under conditions yet to be identified in this and other organisms; consistent with this notion, 3Me-hopanoids contribute to late-stationaryphase survival in Methylococcus capsulatus (18). In vitro, 2Mehopanoids rigidify membranes of varied compositions (12). However, until now, no study has explored whether different hopanoids impact fitness in a natural ecological context.
Recently, it was shown that elimination of hopanoid biosynthesis in photosynthetic Bradyrhizobium strain BTAi1 impairs its symbiosis with the legume Aeschynomene evenia (15). Because the absence of all hopanoids likely had a broad and drastic impact on cellular physiology and hence symbiosis, in this study we tested specific effects of 2Me-and C 35 hopanoids (7) using B. diazoefficiens USDA110, the best-studied Bradyrhizobium strain. In addition to C 30 and C 35 hopanoids (10), B. diazoefficiens makes tetrahymanol, a triterpenoid with a gammacerane skeleton (19) (Fig. 1A; also see Fig. S1 in the supplemental material). Intriguingly, while most hopanoids are thought to occur free within membranes, the C 35 hopanoid, (2-Me) 34-carboxyl-bacteriohopane-32,33-diol, was found to be covalently attached to LPS lipidA, a well-established player in a broad range of host-microbe interactions, to form a compound called hopanoid-lipid A (HoLA) (15,20) (see Fig. S1B).
B. diazoefficiens exhibits two different lifestyles, free living in soil or symbiotic within legume root nodule cells (1,21). In addition to its native soybean host, B. diazoefficiens can engage in nitrogen-fixing symbioses with the stems and roots of the tropical legume Aeschynomene afraspera (22). In both of the these hosts, development of the symbiosis progresses through a series of defined stages: (i) colonization and invasion of host root tissue; (ii) internalization of bacteria by plant cells to form an organelle-like structure called the symbiosome, comprising endosymbiotic bacterial cells termed "bacteroids" that are surrounded by a plantderived "peribacteroid" membrane (see Fig. S2 in the supplemental material); and (iii) initiation of nitrogen fixation by bacteroids, during which there is a high rate of nutrient exchange across the symbiosome membranes between plant-supplied carbon sources and fixed atmospheric nitrogen produced by bacterial nitrogenase (21,23). Supporting such high levels of bacterial nitrogenase activity requires both extensive host control of bacteroid physiology and establishment of a specific host microenvironment, defined by low oxygen, low pH, hyperosmosis, and oxidative stress (24). There is some evidence suggesting that the extent of these environmental stresses varies between plant hosts. For example, A. afraspera was recently shown to produce nodule-specific, cysteine-rich antimicrobial peptides (NCR peptides) that induce differentiation of the bacteroid into an enlarged, elongated, and polyploid state, whereas in the soybean host, NCR peptides are absent and bacteroid morphology and ploidy are similar to those of the free-living state (25). Thus, it is likely that the microbial adaptations required for survival within root nodules are host specific, and we hypothesized that specific hopanoid mutants may exhibit variable phenotypes in diverse plant hosts.
Here, we focus on the phenotypic consequences of the inability of B. diazoefficiens to produce two specific hopanoid classes, 2Meand C 35 hopanoids. We compare its hopanoid-dependent stress phenotypes in the free-living state to those of other hopanoid producers. We further explore the fitness effects of 2Me-or C 35 hopanoid production within a natural ecological context: the symbiotic microenvironment of soybean and A. afraspera cells. These studies begin to define the role of 2Me-and C 35 hopanoids during the progression of plant-microbe symbioses and provide insight into microbial membrane factors that facilitate adaptations to particular microenvironments.

Shc appears to be essential for the survival of B. diazoefficiens.
To eliminate hopanoid production in B. diazoefficiens and to test whether a requirement for hopanoids in efficient symbiosis is conserved between B. diazoefficiens and Bradyrhizobium strain BTAi1, we first attempted deletion of the gene encoding the enzyme catalyzing the first step in hopanoid biosynthesis, squalene hopene cyclase (Shc) (Fig. 1B). We were unable to isolate a ⌬shc mutant using either the pK18mobsacB-based markerless gene deletion method (~400 colonies screened) or the gene replacement strategy with pSUP202pol4 (~1,200 colonies screened) (26). This suggests that Shc might be essential either because hopanoids are required for growth and survival of B. diazoefficiens or because squalene, the substrate of Shc (16), accumulates to toxic levels within the ⌬shc mutant. To rule out the latter possibility, we tried to delete the entire operon encoding squalene-synthesizing enzymes (hpnCDE), shc (hpnF), and hpnG (which catalyzes the second step in the synthesis of C 35 hopanoids) (17), but again, we were unable to obtain the ⌬hpnCDEFG mutant (~150 colonies screened) (Fig. 1B). These results suggest that hopanoid synthesis is essential for the survival of B. diazoefficiens under the conditions used to select the mutants.
C 35 hopanoids contribute to outer membrane rigidity. We employed a fluorescence polarization method by incubating the dye diphenyl hexatriene (DPH) with whole cells to determine whether 2Me-and C 35 hopanoids affect the rigidity of B. diazoefficiens membranes at 25°C and 40°C (Fig. 3). Because previous studies of whole cells of different R. palustris hopanoid mutants indicated that the majority of DPH gets incorporated into the OM, we interpret whole-cell polarization values to reflect the rigidity of the OM (12,29). Membranes of all strains were less rigid at the higher temperature. The ⌬hpnP mutant membrane was as rigid as the WT membrane at both temperatures, whereas the ⌬hpnH mutant membrane was less rigid. Thus, C 35 hopanoids are important for maintaining membrane rigidity in B. diazoefficiens in vivo, in contrast to R. palustris, where the ⌬hpnH mutant membrane showed rigidity similar to that of the WT, despite the capacity of C 35 hopanoids to enhance rigidity in vitro (12). This indicates that the fraction of C 35 hopanoids or HoLA in the OM may be greater in B. diazoefficiens than R. palustris. Despite the lack of C 35 hopanoids, the ⌬hpnH mutant membrane is morphologically indistinguishable from the WT membrane, as seen in whole-cell cryo-transmission electron microscopy (cryo-TEM) micrographs (see Fig. S3 in the supplemental material). C 35 hopanoids are important for aerobic growth. Does a less rigid membrane affect the fitness of ⌬hpnH at different temperatures? To address this question, we compared aerobic growth rates of the ⌬hpnH mutant at 30°C and 37°C with those of the WT and the ⌬hpnP mutant ( Fig. 4A and B). The ⌬hpnP mutant grows like the WT at both temperatures, whereas the ⌬hpnH mutant grows slower at 30°C and is unable to grow at 37°C. These results suggest that C 35 hopanoids are important for growth at ambient temper-  35 hopanoids, such as bacteriohopanetetrol (BHT; R 2 ϭOH) and aminobacteriohopanetriol (aminotriol; R 2 ϭNH 2 ); and tetrahymanol. All these compounds can be methylated at C-2 (2Me, R 1 ϭCH 3 ). (B) Hopanoid biosynthetic gene cluster of B. diazoefficiens. In this study, we focused on the genes colored in gray: the shc (squalene hopene cyclase) product catalyzes squalene cyclization to hopene, the first reaction in the hopanoid biosynthetic pathway; the hpnH product catalyzes addition of adenosine to hopene, the first reaction in the synthesis of C 35 hopanoids; and the hpnP product catalyzes C-2 meth-(Continued) ature (30°C) and essential for growth at higher temperature (37°C). As shown in our whole-cell membrane fluidity measurements, the higher the temperature, the less rigid the membrane. This might be the reason why C 35 hopanoids are absolutely required to maintain membrane rigidity at 37°C but are dispensable at 30°C. It is important to note that the phenotypic defect of the ⌬hpnH mutant could be due to either the absence of C 35 hopanoids or the lack of downstream products, such as HoLA, and even accumulation of the HpnH substrate diploptene, or a combination of these factors. 2Me-and C 35 hopanoids are important for microaerobic growth, and C 35 hopanoids are involved in stress tolerance. Hopanoids have been shown to contribute to stress tolerance in diverse organisms (13)(14)(15)(16). We speculated that such protection would also be seen in B. diazoefficiens. To test this hypothesis, we challenged ⌬hpnP and ⌬hpnH mutants with a variety of stressors that are relevant during the initiation and progression of symbiosis, such as hypoxia, acidic pH, high osmolarity, reactive oxygen species, and peptide antibiotics (24,25).
Under hypoxic conditions with 0.5% oxygen, the ⌬hpnP mutant is unable to attain growth yields as high as those of the WT and the ⌬hpnH mutant fails to grow (Fig. 4C). This indicates that in the free-living state 2Me-hopanoids contribute to microaerobic growth and C 35 hopanoids are essential. Using GC-MS, we determined the abundance of these hopanoid types in the WT (see Table S3 in the supplemental material). The amount of 2Mehopanoids dramatically increased from 33% Ϯ 2% TLE under oxic conditions to 77% Ϯ 2% TLE under hypoxic conditions. This is consistent with hopanoid methylation being important to sustain WT levels of microaerobic growth. The only C 35 hopanoid detectable by GC-MS, BHP-508, increased in abundance from 3% Ϯ 1% TLE for cells grown aerobically to 21% Ϯ 1% TLE when grown microaerobically, in agreement with a microaerobic growth defect for the ⌬hpnH mutant.
Under acidic conditions (pH 6), the ⌬hpnH mutant is unable to grow (Fig. 4D). The ⌬hpnH mutant is also more prone to stationary-phase stress, osmotic stressors (NaCl and inositol), and membrane destabilizers (bile salts and EDTA [17]) than the WT, as evidenced by a reduction in ⌬hpnH mutant growth on stressor gradient plates (Fig. 4E). Additionally, disc diffusion assays showed that the ⌬hpnH mutant is more sensitive to oxidative (hydrogen peroxide [H 2 O 2 ]), detergent (SDS), and acidic (hydrochloric acid [HCl]) stresses than the WT (Fig. 4F). Because B. diazoefficiens is exposed to NCR peptides in A. afraspera, we also tested the sensitivity of the ⌬hpnH mutant to two antimicrobial peptides, polymyxin B (30) and NCR335 from the legume Medicago truncatula (31). The ⌬hpnH mutant displayed a 10-foldlower MIC (48 g/ml) for polymyxin B than did the WT (512 g/ ml). In addition, the ⌬hpnH mutant was found to be 100-fold more susceptible than the WT to NCR335 (Fig. 4G). The ⌬hpnP mutant withstood all of the abovementioned stressors as well as did the WT, with the exception of acidic stress, where it grew slower than the WT. C 35 hopanoids are required to establish an efficient symbiosis with A. afraspera but not with soybean. Because hopanoids are required for microaerobic growth and stress tolerance in the free-living state, we hypothesized that they would aid survival within the plant microenvironment. To test this, we analyzed the  ization show that rigidity decreases for all strains as temperature increases and that the ⌬hpnH mutant membrane is less rigid than that of the WT or the ⌬hpnP mutant (**, P Ͻ 0.01 by Student's two-tailed t test). Error bars represent the standard deviations from three biological replicates (~22 technical replicates). symbiotic phenotypes of ⌬hpnP and ⌬hpnH mutants on two host plants, soybean and A. afraspera. On soybean, at 21 days post inoculation (dpi), both mutants induced fewer nodules and displayed slightly reduced nitrogenase activity as estimated by the acetylene reduction assay (ARA) relative to the WT (see Fig. S4A to C in the supplemental material). However, these differences were not statistically significant, and cytological analyses done by confocal microscopy and TEM revealed no differences between WT and mutant nodules (see Fig. S4M to X). Thus, under the conditions of these assays, neither 2Me-nor C 35 hopanoids appear to be important for symbiosis between B. diazoefficiens and soybean.
In contrast, a clear effect of the hpnH mutation could be observed when B. diazoefficiens infected A. afraspera. Plants inoculated with the ⌬hpnH mutant displayed typical nitrogen starvation symptoms, including foliage chlorosis, reduced plant growth, and half the acetylene reduction activity of WT-and ⌬hpnP mutant-infected plants ( Fig. 5A and B). Reduced nitrogen fixation was not due to a decrease in the numbers of nodules, which were comparable in WT-and ⌬hpnH mutant-infected plants at 9, 14, and 21 dpi, suggesting that the hpnH mutation does not affect nodule organogenesis ( Fig. 5C; see also Fig. S5 in the supplemental material). However, cytological analyses revealed that ⌬hpnH mutant nodules displayed several disorders in comparison to WT and ⌬hpnP mutant nodules (Fig. 5D to Z=).
At the cellular level, ⌬hpnH mutant nodules were smaller (Fig. 5D to F) and had pink or, in~30% of cases, even white central tissue in contrast to WT and ⌬hpnP mutant nodules, which were dark pink due to the accumulation of the O 2 carrier leghemoglobin ( Fig. 5G to I). The central symbiotic tissue of ⌬hpnH mutant nodules was often disorganized and partially infected (Fig. 5L, M, and R), as opposed to the fully occupied tissue of WT and ⌬hpnP mutant nodules (Fig. 5J, K, P, and T). In some ⌬hpnH mutant nodules, the presence of necrotic regions-characterized by the accumulation of autofluorescent brown compounds-could be seen ( Fig. 5M and N). These are likely polyphenol compounds whose production is associated with plant defense responses (32). Within ⌬hpnH mutant nodules, iodine staining also revealed accumulation of starch granules in the noninfected cells surrounding the symbiotic tissue, whereas such granules were rarely observed in WT and ⌬hpnP mutant nodules (Fig. 5O). Starch accumulation is indicative of an imbalance between the photosynthates furnished by the plant and the ability of the bacterium to metabolize them, a typical feature of nonfixing or underperforming strains (33,34).
To determine whether ⌬hpnH mutant symbiotic defects stem from a problem in the bacterial differentiation process or are due to a damaged membrane, we examined nodule sections by confocal microscopy using live-dead staining (35) and TEM to analyze the ultrastructure of bacteroids. Confocal microscopy revealed that all strains, including the ⌬hpnH mutant, differentiated properly into elongated bacteroids, which were, for the majority, viable, as indicated by the green Syto9 staining (Fig. 5P to U). However, TEM analysis showed that the cell envelope of some ⌬hpnH mutant bacteroids was not well delineated and, in a few cases, even broken ( Fig. 5Y and Z=). Similar damage was seen in the peribacteroid membrane that surrounds bacteroids. Deposits of cellular material, possibly resulting from the release of plant or bacterial cytoplasm, were also observed in the peribacteroid space, suggesting a beginning of senescence or perhaps necrosis of symbiotic  bacterial cells (Fig. 5Z and Z=). Such defects were not observed in the WT (Fig. 5V to X) or ⌬hpnP nodules. Taken together, our data indicate that under these conditions, C 35 hopanoids, but not 2Mehopanoids, play an important role in facilitating the fitness of B. diazoefficiens in symbiosis with A. afraspera.

DISCUSSION
Bacteria that provide plants with fixed nitrogen represent an attractive agronomical alternative to nitrogen fertilizers (36). Recently, we found a statistically significant correlation between the presence of hopanoid biosynthetic genes and organisms, metabolisms, and environments known to support plant-microbe interactions (9). This raised the hypothesis that hopanoids could support bacterial fitness in the context of symbiosis for certain organisms. Building on our recent observation that hopanoids promote symbiosis between the legume A. evenia and the Bradyrhizobium BTAi1 strain (15), we explored the generality of this finding by studying the phylogenetically different and betterknown Bradyrhizobium species B. diazoefficiens, focusing on the roles of two dominant hopanoid classes. In the free-living state of B. diazoefficiens, 2Me-hopanoids contribute to growth under microaerobic and acidic conditions and C 35 hopanoids are required for microaerobic growth and tolerance to diverse stresses found in the symbiotic microenvironment. Consistent with these phenotypes, C 35 hopanoids are critical for symbiosis between B. diazoefficiens and A. afraspera, and yet they are dispensable for symbiosis with soybean. This intriguing finding suggests that the microenvironment encountered by plant symbionts varies between hosts.
Bradyrhizobium strain BTAi1 and B. diazoefficiens differ physiologically in important ways. Shc proteinss from these two strains fall in distinct phylogenetic clades (15). Unlike Bradyrhizobium strain BTAi1, B. diazoefficiens is unable to photosynthesize (37,38). Moreover, B. diazoefficiens infects plants via a Nod factordependent pathway, whereas Bradyrhizobium strain BTAi1 uses alternate symbiotic strategies (39). Our inability to delete shc in B. diazoefficiens suggests that hopanoids are essential in this species, in contrast to Bradyrhizobium strain BTAi1, where shc mutants are viable. This fundamental difference between the species likely reflects differences in the niches that they inhabit as a consequence of their metabolic differences and what is required for survival therein.
What insights can we gain about 2Me-and C 35 hopanoid functions in natural contexts based on ex planta experiments? This is the first study to identify conditions under which synthesis of 2Me-hopanoids is important for any cell type. These include hypoxic and acidic conditions that B. diazoefficiens possibly perceives as stress, thus upregulating 2Me-hopanoid production as has been seen in R. palustris (13) to enhance membrane rigidity and stability (12). However, it is puzzling that 2Me-hopanoids are dispensable under similar conditions within the plant cell, and a priority for future work will be to explain this paradox. Prior work in B. cenocepacia has shown that C 35 hopanoids promote stress tolerance and antibiotic resistance (14). Similarly, we found that synthesis of C 35 hopanoids is important for growth under oxic and hypoxic conditions and for tolerance to diverse stressors. Consistent with our whole-cell membrane rigidity measurements, these phenotypes could be due to higher membrane fluidity resulting from the absence of C 35 hopanoids, the inability to make HoLA, and/or the accumulation of the C 30 hopanoid diploptene. We hope to tease apart these possibilities going forward. Intriguingly, the abundance of 2Me-hopanes in ancient sediments peaks during oceanic anoxic events (8), and today, C 35 hopanoids appear to be enriched in hypoxic regions of the oceans (40).
Why do we observe context-dependent hopanoid phenotypes in planta? The phenotypic difference of the requirement for C 35 hopanoids in A. afraspera but not in soybean likely stems from differences between the plants' intracellular environments. In both plants, the bacterium is exposed to a variety of stresses, including oxidative, osmotic, and acidic stresses, within the microaerobic niche of the infected plant cell (24). Although such an environment is less than ideal for the ⌬hpnH mutant, it is able to colonize as evidenced by its successful symbiosis with the soybean plant. However, unlike soybean, in A. afraspera, B. diazoefficiens undergoes terminal differentiation due to the action of NCR peptides (25). Because the ⌬hpnH mutant is highly sensitive to NCR peptides, exposure within the host might reduce the viable mutant population by causing cell death or increasing susceptibility to this and other plant defense mechanisms. Consistent with this hypothesis, the ⌬hpnH mutant only partially infects the A. afraspera nodule tissue.
In A. afraspera, synthesis of C 35 hopanoids is critical for several aspects of the symbiosis, including evasion of plant defense reactions, efficient utilization of plant photosynthates, and nitrogen fixation. Two reasons why the plant host mounts an immune response against the ⌬hpnH mutant may be that the altered mutant surface layer, as seen in TEM images, is unable to suppress this response (41) and/or the host induces nodule senescence prematurely on detecting an underproductive symbiont (42). Consistent with this, nitrogenase activity is reduced in the ⌬hpnH mutant relative to the WT, a likely consequence of poor cell viability. Similarly, the buildup of plant carbon as starch in ⌬hpnH mutant nodules might indicate slow metabolism and/or perturbation of membrane transport processes that facilitate bacteroid carbon acquisition.
The global agricultural economy is largely based on nitrogen fertilizers, with the United States alone consuming 13,000 tons per annum (43). However, the usage of nitrogen fertilizers comes with a price, as their production requires burning of fossil fuels and their runoff from soils leads to surface and groundwater contamination (44). Rhizobia, which naturally fix nitrogen in association with common crops, are an environmentally and economically feasible alternative to nitrogen fertilizers. Our results reveal that hopanoids affect the ability of B. diazoefficiens to cope with environmental stresses as well as its nitrogen fixation efficiency in a plant host-dependent manner. This observation is relevant to interpreting ancient patterns of hopane deposition, which correlate with paleoenvironmental conditions where nitrogen fixation may have provided a selective advantage (8). In addition, understanding the roles of hopanoids in bacterial stress resistance and how they facilitate nitrogen fixation may enable the engineering of agronomically useful strains with enhanced tolerance to rising temperature and salinity.

MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains used in this study are listed in Table S4 in the supplemental material. Escherichia coli strains were grown in lysogeny broth (LB) (45) at 37°C. B. diazoefficiens strains were grown at 30°C in either rich medium (peptone-salts-yeast extract medium with 0.1% arabinose [PSY] [46] or yeast extract-mannitol medium [YM] [47]) or minimal medium (buffered nodulation medium [BNM] [48]).
DNA methods, plasmid construction, and transformation. All plasmid constructions and primers used in this study are described in Table S4 in the supplemental material. Standard methods were used for plasmid DNA isolation and manipulation in E. coli (49,50). DNA sequences of all cloning intermediates were confirmed by sequencing at Retrogen (San Diego, CA). Plasmids were mobilized by conjugation from E. coli S17-1 into B. diazoefficiens strains as previously described (51).
Hopanoid analysis. Lipid extraction was performed by the Bligh-Dyer method followed by hopanoid analysis using high-temperature GC-MS (16) and ultraperformance LC-MS (29) as previously described. Growth of bacterial cultures and analysis methods have been described in detail in Text S1 in the supplemental material.
LipidA analysis. Isolation, purification, and characterization of lipidA from B. diazoefficiens strains were performed by gas-liquid chromatography-MS (GLC-MS) and MALDI-time of flight (TOF) MS as described in reference 15 and in Text S1 in the supplemental material.
Membrane rigidity. For whole-cell membrane rigidity measurements, we used the fluorescence polarization method using the dye DPH described in reference 12.
Growth curves, stress assays, and MIC determinations. The growth curve assays, stress assays, and MIC determinations were performed as outlined in references 13 and 15 and have been described in detail in Text S1 in the supplemental material.
Plant cultivation, symbiotic analysis, and microscopy. Soybean and A. afraspera plants were grown hydroponically as described in reference 22. The plant infection assays and cytological analyses methods are outlined in Text S1 in the supplemental material.