Putative type III effector SkP48 of Bradyrhizobium sp. DOA9 encoding a SUMO protease blocks nodulation in Vigna radiata

Abstract

Bradyrhizobium sp. DOA9 can nodulate a wide spectrum of legumes; however, unlike other bradyrhizobia, DOA9 carries a symbiotic plasmid harboring type III secretion system (T3SS) and several effector (T3E) genes, one of which encodes a putative type III effector SkP48. Here, we demonstrated the pivotal roles of SkP48 from Bradyrhizobium sp. DOA9 in inhibiting nodulation of various Vigna species and Crotalaria juncea and suppressing nodulation efficiency of Arachis hypogaea. By contrast, the nodulation efficiency of a SkP48 mutant did not differ significantly with the DOA9 wild-type strain on Macroptilium atropurpureum and Stylosanthes hamata. The SUMO domain of SkP48 is primarily responsible for the blocking nodulation phenotype V. radiata. An evolutionary analysis revealed that the SkP48 which contains a shikimate kinase and a SUMO protease (C48 cysteine peptidase) domain, SkP48 is distinct from other effectors previously reported in other bradyrhizobia and pathogenic bacteria. Our findings suggest that the putative T3E SkP48 is a key factor suppressing nodulation and nodule organogenesis in several legumes by activation of effector-triggered immunity through salicylic acid biosynthesis induction, which is deleterious to rhizobial infection. In addition, nodulation may be modulated by the function of defensins involved in jasmonic acid signalling in V. radiata SUT1.

Keywords: Bradyrhizobium sp. DOA9; Vigna radiata; SkP48; Symbiosis; T3SS.

Fig. 1

Schematic representation of the putative effector protein SkP48 harboring the shikimate kinase and C48 SUMO protease domains in DOA9 (A). (B) Phylogenetic analysis of SkP48 and their homologs among rhizobia was constructed using the Maximum Likelihood method. The analysis used the Jones-Taylor-Thornton (JTT) and Gamma Distributed (G) models, with 1,000 bootstrap replications. (C) qRT-PCR expression of putative T3E SkP48 in Bradyrhizobium sp. DOA9 (DOA9) and ttsI (T3SS regulator) mutant strain (ΩttsI) were determined by qRT-PCR in presence or absence of genistein inducer. Data are presented as means ± standard deviations. Different letters indicate significant differences at P < 0.05.

Fig. 2

Nodulation of Bradyrhizobium sp. DOA9 and its mutant derivatives in Vigna radiata cv. SUT1. Cytological aspect of nodules (A-C) at 10 dpi: A (DOA9), B (ΩrhcN), and C (ΔSkP48); (D-F) 15 dpi: D (DOA9), E (ΩrhcN), and F (ΔSkP48); and (G-I) 30 dpi: G (DOA9), H (ΩrhcN), and I (ΔSkP48). Bar = 1 mm. Propidium iodide (PI) and SYTO9 staining of sectioned nodules (J-L): J (WT), K (ΩrhcN), and L (∆SkP48). Total nodule number per plant (M), ARA activity (N), and plant dry weight (O) were determined at 30 dpi. “*”, P < 0.05 and “**”, P < 0.01 according to Student’s t-test. The means followed by different letters are significantly different at the 5% level (P ≤ 0.05 according to Tukey’s test) (n = 6). Nodulation of Bradyrhizobium sp. DOA9 and its mutant derivatives in Vigna mungo. Cytological aspect of root nodules (P-R): P (DOA9), Q (ΩrhcN), and R (ΔSkP48). Bar = 1 mm. Propidium iodide (PI) and SYTO9 staining of sectioned nodules (S-U): S (WT), T (ΩrhcN), and U (∆SkP48). Total nodule number per plant (V), ARA activity (W), and plant dry weight (X) were determined at 30 dpi. The means followed by different letters are significantly different at the 5% level (P ≤ 0.05 according to Tukey’s test) (n = 6).

Fig. 3

Nodulation of Bradyrhizobium sp. DOA9 and its mutant derivatives in Crotalaria juncea. Cytological aspect of roots (A-C): A (DOA9), B (ΩrhcN), and C (ΔSkP48); bar = 1 cm. Nodule morphology (D-F): D (DOA9), E (ΩrhcN), and F (ΔSkP48). Bar = 1 mm. Propidium iodide (PI) and SYTO9 staining of sectioned nodules (G-I): G (WT), H (ΩrhcN), and I (∆SkP48). Total nodule number per plant (J), ARA activity (K) and, plant dry weight (L) were determined at 30 dpi. The means followed by different letters are significantly different at the 5% level (P ≤ 0.05 according to Tukey’s test) (n = 6).

Fig. 4

Nodulation of Bradyrhizobium sp. DOA9 and its mutant derivatives in Arachis hypogea. Cytological aspect of roots (A-C): A (DOA9), B (ΩrhcN), and C (ΔSkP48). Bar = 1 cm. Nodule morphology (D-F): D (DOA9), E (ΩrhcN), and F (ΔSkP48). Bar = 1 mm. Propidium iodide (PI) and SYTO9 staining of sectioned nodules (G-I): G (WT), H (ΩrhcN), and I (∆SkP48). Total nodule number per plant (J), ARA activity (K) and, plant dry weight (L) were determined at 30 dpi. The means followed by different letters are significantly different at the 5% level (P ≤ 0.05 according to Tukey’s test) (n = 6).

Fig. 5

Nodulation phenotypes of Bradyrhizobium sp. DOA9 and its derivatives (Δp0490, Δp0871, Δp0903, ΔSkP48::p0903 (ΔSkP48 complement with p0903-SUMO-lack), ΔSkP48::p0903 + SkP48-SK (ΔSkP48 complement with p0903-SkP48-SK), and ΔSkP48::p0903 + SkP48-SUMO (ΔSkP48 complement with p0903-SkP48-SUMO) on the V. radiata SUT1. (A) Schematic representation of DOA9 and its complement derivatives strains. ΔSkP48 complement with shikimate kinase (SK) and SUMO protease (a ubiquitin-like protease: ULP) domain. Evaluation at 30 dpi included (B) nodule count per plant, means followed by different letters are significantly different at the 1% level (P ≤ 0.01, according to Tukey’s test), with a sample size of n = 3. Nodule morphology in V. radiata SUT1 (scale bar: 1 mm). Nodule sections were stained with SYTO9 (living bacteroid cells: green), propidium iodide (dead bacteroid cells: red), and calcofluor (plant cell walls: blue); scale bar for confocal images: 100 μm.

Fig. 6

qRT-PCR results of gene expression in Vigna radiata cv. SUT1 inoculated with DOA9 and ΔSkP48 at 4 dpi. (A) Expression levels of differentially expressed genes in SUT1: chalcone synthase (CHS), non-disease resistance 1 (NDR1), pathogenesis-related genes transcriptional activator 5 (PTi5), pathogenesis-related genes transcriptional activator 6 (PTi6), pathogenesis-related protein 2 (PR2), and pathogenesis‐related protein 5 (PR5). (B) Salicylic acid content of V. radiata roots: non-inoculation (NI), DOA9 inoculation (DOA9), and SkP48 mutant strain (ΔSkP48) inoculation. Results were derived from triplicate experiments. Asterisks indicate significant differences at P < 0.05.