Identification and functional analysis of a type III effector of Bradyrhizobium elkanii that hijacks symbiosis signaling in soybean

概要

Introduction
Legume plants compensate for soil nitrogen deficiency by establishing a root-nodule symbiosis with nitrogen-fixing bacteria (rhizobia) in exchange for host-plant carbon supply. This mutualistic interaction is highly specific, and mostly relying on rhizobium-produced lipochitooligosaccharides (Nod factors; NFs) and their perception by leguminous receptors (NFRs), which activate nodulation signaling. Unexpectedly, soybean microsymbiont Bradyrhizobium elkanii utilizes the type III secretion system (T3SS), which is known as a common infection strategy employed by many pathogenic bacteria for delivering their virulence proteins (effectors), to hijack leguminous nodulation signaling. The present study is to elucidate the molecular basis of NF-independent T3SS-dependent nodulation in legume-rhizobium symbiosis.

Objective
1. Identification and characterization of a Bradyrhizobium elkaniii USDA61 T3 effector that determines NF-independent symbiosis with soybean
2. Transcriptome analysis of soybean genes in NF-independent symbiosis by Bradyrhizobium elkanii USDA61 T3 effector
3. Functional analysis of Bradyrhizobium elkanii USDA61 T3 effector that triggers NF-independent symbiosis with soybean

Methods, results, and discussion
1. Identification of the responsible T3 effector triggering NF-independent nodulation
To identify the T3 effector triggering NF-independent nodulation, the T3 effector candidates were screened from B. elkanii genome by in-silico analysis. Following, mutation of B. elkanii genes putatively encoding T3 effectors, and inoculation of soybean En1282 (nfr1 mutant) with constructed mutants were conducted. Among the T3 effector candidates, the Bel2-5 was confirmed to be responsible for triggering NF-independent T3SS-dependent nodulation based on the observation that B. elkanii wild-type, but not the bel2-5 deletion mutant was formed nitrogen-fixing nodules on soybean nfr1
mutant. The Bel2-5 does not appear to be rhizobium-specific, as it shares similarity with XopD effector from plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv). Taken together, these results suggest that Bel2-5 is a pathogen-like effector adopted by B. elkanii that hijacks soybean nodulation signaling in NF-independent manner.

2. Transcriptional analysis of soybean genes controlled by Bel2-5 effector
Molecular signaling caused by B. elkanii Bel2-5 to trigger NF-independent T3SS-dependent nodulation in soybean nfr1 mutant was investigated using qRT-PCR and RNA-seq analyses. Comparative transcriptional analysis of soybean roots inoculated with B. elkanii wildtype and bel2-5 deletion mutant showed that Bel2-5 was able to activate nodulation-specific genes, ENOD40 and NIN that were previously thought to be up-regulated only following the recognition of NFs via associated NFRs. Furthermore, Bel2-5 was induced the expression of cytokinin biosynthesis-related genes (IPT5, CYP735A, and LOG1-like) on the root of soybean nfr1 mutant. In contrast, ethylene biosynthesis gene (ACO1) and plant defense-related genes (WRKY33/75, ERF1b, and ERF98) were down-regulated by the presence of Bel2-5. These results suggest that B. elkanii Bel2-5 likely affects cytokinin levels in the nfr1 mutant soybean, leading to nodule development.

3. Functional analysis of Bel2-5 domains defines nodulation ability in NF-independent manner
Functional analysis of Bel2-5 domains was conducted using in-silico analysis, mutational analysis, heterologous expression in yeast cells, and domain-swapped chimaeras between Bel2-5 and Xcv XopD. The results showed that Bel2-5 is a modular protein that possesses multiple domains, including two internal repeat domains, two ethylene (ET)-responsive element- binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain which are confirmed to be essential for Bel2-5 activity in triggering nodulation and heterologous expression in yeast cells. Furthermore, functional analysis on species-specific between B. elkanii Bel2-5 and Xcv XopD by ULP domain-swapped chimaeras confirmed that C-terminal of these effectors are partially exchangeable and may function similarly during nodulation, while other parts of Bel2-5 also contain unique features that are involved in the differentiation of species-specific functions in the respective host plants.

Conclusion
A recent study identified rhizobia use a pathogen-like effector Bel2-5 to hijack soybean nodulation signaling in NF-independent manner. The Bel2-5 promotes expression of host-cytokinin related genes, which favourable for nodule organogenesis, while suppressing ethylene-defense and related genes, which are detrimental to nodulation. Bel2-5 carrying multiple domains confirmed necessary for symbiosis. These findings indicate that Bel2-5 evolved to perform a symbiotic function during coevolution with host legume and could pave the way for transferring nitrogen-fixing nodulation into cereal crops.