Bradyrhizobium diazoefficiens Requires Chemical Chaperones To Cope with Osmotic Stress during Soybean Infection

Abstract

When engaging in symbiosis with legume hosts, rhizobia are confronted with environmental changes, including nutrient availability and stress exposure. Genetic circuits allow responding to these environmental stimuli to optimize physiological adaptations during the switch from the free-living to the symbiotic life style. A pivotal regulatory system of the nitrogen-fixing soybean endosymbiont Bradyrhizobium diazoefficiens for efficient symbiosis is the general stress response (GSR), which relies on the alternative sigma factor σEcfG. However, the GSR-controlled process required for symbiosis has not been identified. Here, we demonstrate that biosynthesis of trehalose is under GSR control, and mutants lacking the respective biosynthetic genes otsA and/or otsB phenocopy GSR-deficient mutants under symbiotic and selected free-living stress conditions. The role of trehalose as a cytoplasmic chemical chaperone and stress protectant can be functionally replaced in an otsA or otsB mutant by introducing heterologous genetic pathways for biosynthesis of the chemically unrelated compatible solutes glycine betaine and (hydroxy)ectoine. Alternatively, uptake of exogenously provided trehalose also restores efficient symbiosis and tolerance to hyperosmotic and hyperionic stress of otsA mutants. Hence, elevated cytoplasmic trehalose levels resulting from GSR-controlled biosynthesis are crucial for B. diazoefficiens cells to overcome adverse conditions during early stages of host infection and ensure synchronization with root nodule development. IMPORTANCE The Bradyrhizobium-soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited. In this study, we show that the requirement of the rhizobial general stress response (GSR) during host infection is attributable to GSR-controlled biosynthesis of trehalose. Specifically, trehalose is crucial for an efficient symbiosis by acting as a chemical chaperone to protect rhizobia from osmostress during host infection.

Ensifer aridi LMR001T Symbiosis and Tolerance to Stress Do Not Require the Alternative Sigma Factor RpoE2

“…We found that the mutation resulted in an increased transcription of four genes, while it reduced the expression of three genes. An increased transcription of otsB was obtained in the ∆rpoE2 strain, indicating that RpoE2-mediated GSR in E. aridi involves the T6P pathway, as shown in S. meliloti [45] or in B. diazoefficiens [74]. We also found that for rsiA1, the anti-sigma factor that is located upstream of and overlaps the 5 end of the rpoE2 gene forming an operon was less expressed in the mutant, which suggests, again, that, as shown in other alphaproteobacteria [65], rpoE2 is autoregulated in E. aridi.…”

“…Furthermore, recent work on B. diazoefficiens showed that ecfG mutation resulted in increased sensitivity to various stresses including hyperosmotic and ionic stresses [74]. More interestingly, in both Bradyrhzobium diazoefficiens and Sinorhizobium meliloti, a rpoE2 mutation was shown to repress trehalose 6 Phosphate pathway, which was involved in hyperosmotic stresses' tolerance [45] and was sufficient to impair early symbiotic development in the more sensitive B. diazoefficiens species [74], further supporting that trehalose is a key compatible solute for Bradyrhizobium stress adaptation and nodulation, as shown in B. japonicum [46]. The absence of a symbiotic phenotype observed in the rpoE2 mutant of S. meliloti may result from a high tolerance to hyperosmotic stress in this species as compared to the Bradyrhizobium species.…”

“…The absence of a symbiotic phenotype observed in the rpoE2 mutant of S. meliloti may result from a high tolerance to hyperosmotic stress in this species as compared to the Bradyrhizobium species. Such stress may be encountered, notably, in infection threads, which is supported by the restoration of symbiosis by addition of exogenous trehalose in B. diazoefficiens ∆otsA strain still capable to import it or in a strain capable to endogenously synthesize other compatible osmoprotectants [74]. To explore the function of RpoE2 and its possible role in the regulation of several functions shown to be transcriptionally regulated by salt stress as well as in the tolerance of the LMR001 bacterium to various stresses and symbiosis development, a deletion mutant of the rpoE2 gene (∆rpoE2) was generated and phenotypically characterized.…”

Effect Carrier Materials of Bradyrhizobium sp. strain PZS_A08 on Growth of Indigofera zollingeriana

“…This size is smaller than the Bradyrhizobium nodules in general, which have a length ranging from 5-10 mm for Indigofera (Bünger et al 2021). Nodulations in other plants such as shrubby sophora (Sophora flavescens) and soybean (Glycine max) are smaller, about 1-3 mm long (Ledermann et al 2021;Liu et al 2018). This size is also influenced by the type and age of the plant, where Indigofera has a relatively long life.…”

Maintaining osmotic balance in legume nodules

“…Biosynthesis of compatible solutes such as trehalose or N -acetylglutaminylglutamine amide (NAGGN) contributes to adaptation to high osmolarity environments in Sinorhizobium ( Flechard et al , 2010 ; Sagot et al , 2010 ), Rhizobium ( McIntyre et al , 2007 ), or Bradyrhizobium ( Ledermann et al , 2021 a ). Whilst trehalose biosynthesis is needed in Bradyrhizobium for growth in infection threads ( Ledermann et al, 2018 , 2021 a ), no mutants in compatible solute biosynthesis with an effect on bacteroids have so far been reported. This raises the question of how rhizobia adapt to the osmotic conditions in nodules.…”