The Type III Effectome of the Symbiotic Bradyrhizobiumvignae Strain ORS3257.

Nicolas Busset,Alicia Camuel,Joël Fardoux,Peter Mergaert,Djamel Gully,David Cornu,Eric Giraud,Dany Severac,Albin Teulet

doi:10.3390/biom11111592

Abstract

Many Bradyrhizobium strains are able to establish a Nod factor-independent symbiosis with the leguminous plant Aeschynomene indica by the use of a type III secretion system (T3SS). Recently, an important advance in the understanding of the molecular factors supporting this symbiosis has been achieved by the in silico identification and functional characterization of 27 putative T3SS effectors (T3Es) of Bradyrhizobium vignae ORS3257. In the present study, we experimentally extend this catalog of T3Es by using a multi-omics approach. Transcriptome analysis under non-inducing and inducing conditions in the ORS3257 wild-type strain and the ttsI mutant revealed that the expression of 18 out of the 27 putative effectors previously identified, is under the control of TtsI, the global transcriptional regulator of T3SS and T3Es. Quantitative shotgun proteome analysis of culture supernatant in the wild type and T3SS mutant strains confirmed that 15 of the previously determined candidate T3Es are secreted by the T3SS. Moreover, the combined approaches identified nine additional putative T3Es and one of them was experimentally validated as a novel effector. Our study underscores the power of combined proteome and transcriptome analyses to complement in silico predictions and produce nearly complete effector catalogs. The establishment of the ORS3257 effectome will form the basis for a full appraisal of the symbiotic properties of this strain during its interaction with various host legumes via different processes.

Highlights

Bradyrhizobia are Gram-negative bacteria that are widespread and dominant in soils all around the world [1]
The expression of the T3SS machinery and T3SS effectors (T3Es) is under the control of the TtsI transcriptional regulator, which is in turn induced by the NodD transcriptional activator and plant-derived flavonoids [24]
This requires, for each strain, identifying the cocktail of effectors secreted since certain effectors can act in synergy, have functional redundancies, or have opposing effects on the symbiotic process [23,38,39]

Summary

Introduction

Bradyrhizobia are Gram-negative bacteria that are widespread and dominant in soils all around the world [1]. The best-known Bradyrhizobium strains are symbiotic nitrogen fixers, interacting with a large diversity of legumes, including plants of agronomic interest [2] This symbiosis leads to the formation of new root organs, the nodules, in which the bacteria differentiate into a nitrogen-fixing form called bacteroids. Bacteria obtain a privileged ecological niche within the nodules where they can massively multiply thanks to nutrients provided by the plant, including reduced carbon resulting from photosynthesis. This symbiotic process between the two partners is regulated by the exchange of signal molecules. One of the key steps allowing the initiation of symbiosis is the secretion by the bacteria of lipochitooligosaccharide signals called Nod factors (NFs), whose synthesis

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