Abstract
Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, 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 all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii, which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean.
Highlights
Legume plants cope with nitrogen-deficient soils by establishing symbiosis with nitrogen-fixing bacteria, called rhizobia
The nodule numbers observed with the other mutants, namely, BERD1, BERD2, BEC1286A, and BEEAR2, were decreased to similar levels as those observed upon inoculation with the bel2-5 deletion mutant
These results demonstrate that some domains/motifs (RD1, repeat domain 2 deletion mutant (RD2), C1286A, and amino acid substitution mutant of the EAR2 (EAR2)) play pivotal roles in Bel2-5 during nodulation of the nfr1 mutant soybean, while amino acid substitution mutant of the EAR1 (EAR1) and nuclear localization signal (NLS) are less important
Summary
Legume plants cope with nitrogen-deficient soils by establishing symbiosis with nitrogen-fixing bacteria, called rhizobia. This symbiosis results in the formation of a unique organ, the root nodule, where rhizobia provide ammonia for plants in exchange for carbon sources and other nutrients essential for their growth (Oldroyd et al, 2011). This symbiosis is of agronomic importance, as it reduces the need for chemical fertilizers in the production of legume crops such as soybean (Soumare et al, 2020). Most remarkably, numerous rhizobia utilize a type III secretion system (T3SS), which in pathogenic bacteria is known for its delivery of virulence factors, to promote symbiosis with host legumes (Deakin and Broughton, 2009)
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