A new type III effector from Bradyrhizobium sp. DOA9 encoding a putative SUMO-protease blocks nodulation in Arachis hypogaea L

Abstract

Effector proteins secreted via the type III secretion system (T3SS) of nitrogen-fixing rhizobia are key determinants of symbiotic compatibility in legumes. Previous report revealed that the T3SS of Bradyrhizobium sp. DOA9 plays negative effects on Arachis hypogaea symbiosis. In this study, we characterized the symbiotic role of 4 effector proteins (p0490, p0871, SkP48, and p0903) containing the small ubiquitin-like modifier (SUMO) protease domain identified in DOA9 during symbiosis. While the DOA9 strain and the two mutants of SUMO-proteases, p0490 and p0871, induced inefficient nodulation in A. hypogaea, the mutation of SUMO-proteases SkP48 or p0903 promoted efficient symbiosis comparable to the type strain Bradyrhizobium arachidis CCBAU051107. Complementation study of ∆p0903 with various mutated forms of p0903 highlighted importance of ubiquitin-like protein (ULP) domain in restriction of nodulation in A. hypogaea. We observed the accumulation of jasmonic acid (JA) and upregulation of several defence genes involved in the JA/ethylene (ET) signalling pathway at the early stage of infection in roots inoculated with DOA9 strain compared with those inoculated with the DOA9-∆p0903 strain. Our data highlight the importance of SUMO-protease effectors during the symbiotic interaction between bradyrhizobia and A. hypogaea, which could be useful for the development of high-performance inocula to improve its growth.

Keywords: Arachis hypogaea; Bradyrhizobium sp; T3SS; and SUMO-protease.

Fig. 1

Phylogenetic tree analysis of four the SUMO-proteases identified in Bradyrhizobium sp. DOA9 and other SUMO-proteases from Bradyrhizobium species isolated from 6 legume crops indicated in the key. XopD from Xanthomonas campestris was used as an outgroup for tree rooting. The GenomeScope and NCBI-derived amino acid sequences of SUMO-proteases were subjected to phylogenetic analyses using IQ-tree v2.2.0.3. The phylogeny was inferred using the maximum-likelihood method, and branch supported with ultrafast bootstrap from 150,000 iterations. The SUMO-proteases from DOA9 were indicated with black dots.

Fig. 2

The structure organization of four-effector proteins containing the small ubiquitin-like modifier (SUMO) protease domain in DOA9 compared with other putative SUMO-proteases among Bradyrhizobium species and XopD from pathogenic bacteria Xanthomonas campestris. The domain organization includes the repeat domain (blue box), shikimate kinase domain (red box), and ubiquitin-like protease domain (orange box) are shown.

Fig. 3

The symbiotic nodulation of Bradyrhizobium sp. DOA9 and its derivative mutants (DOA9-ΩrhcN, DOA9-∆p0490, DOA9-∆p0871, DOA9-∆p0903, and DOA9-∆SkP48), as well as Bradyrhizobium arachidis LMG26795 and its T3SS mutant (LMG26795-ΩrhcN) derivative in peanut (KK5). The assessment at 30 dpi included (a) nodule number per plant, (b) ARA activity, and (c) plant dry weight. The means followed by different letters are significantly different at the 0.1% level (P ≤ 0.001, according to Tukey’s test), with a sample size of n = 10, except for ARA activity, where n = 5. (d) Nodulation in peanut (KK5) manifested as nodules morphology (scale bar: 100 mm), and the nodule sections were stained with SYTO9 (living bacteroid cells: green), propidium iodide (dead bacteroid cells: red), and calcofluor (plant cell wall: blue); scale bar for confocal image: 200 and 50 μm.

Fig. 4

The symbiotic nodulation of Bradyrhizobium sp. DOA9 and its derivative mutants (DOA9-ΩrhcN, DOA9-Δp0903, and Δp0903 + p0903) inoculated in various peanut cultivars (KK5, TN9, KS2, and SK38). (a-d) The nodule numbers per plant of peanut cultivars (KK5, TN9, KS2, and SK38, respectively) at 30 dpi. The means followed by different letters are significantly different at the 0.1% level (P ≤ 0.001, according to Tukey’s test, with a sample size of n = 6).

Fig. 5

Nodulation phenotypes of Bradyrhizobium sp. DOA9 and its derivatives (DOA9-ΩrhcN, DOA9-Δp0903, Δp0903 + p0903, Δp0903 + p0903-ULP-lack, Δp0903 + p0903-D728A, and Δp0903 + p0903-D763A) on the peanut cultivar (KK5). (a) Schematic representation of DOA9 and its mutant derivatives strains. SUMO-protease p0903 possesses two internal repeat domains, a ubiquitin-like protease (ULP) domain, and catalytic core amino acids aspartic acid (D728A) and cysteine (C763A) in ULP domain. Evaluation at 30 dpi included (b) nodule count per plant, (c) ARA activity, (d) chlorophyll content, and (e) plant dry weight. The 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 = 10, except for ARA activity, where n = 5. (f) Nodulation in peanut (KK5) manifested as nodule morphology (scale bar: 200 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 and jasmonic acid levels in peanut roots (KK5). The relative expression levels of genes in peanut roots inoculated with DOA9 and DOA9-Δp0903 at 3 dpi are shown for: (a) symbiotic-related genes (cyclic nucleotide-gated channels [CNGC], symbiotic remorins [SYMREM], and E3 ubiquitin-protein ligase LIN-1 [CERBERUS]); (b) jasmonic acid (JA)-related genes (transcription factor [Myc2], pathogenesis-related protein 4 [PR4], and defensin [Def2-2]). The means followed by different letters are significantly different at the 5% level (P ≤ 0.05, according to Student’s t-test, with a sample size of n = 3). (c) Jasmonic acid concentrations in peanut roots for the following treatments: Non-Inoculated (NI), DOA9, DOA9-Δp0903, and Δp0903 + p0903-ULP-lack at 3 dpi. The 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).

Fig. 7

Schematic overview of the putative SUMO-protease p0903 T3E symbiotic process between Bradyrhizobium sp. DOA9 and peanut (KK5). Theoretically naringenin induces the expression level of the putative T3E-p0903 gene. Consequently, SUMO-protease p0903 might trigger phytohormone-mediated effector-triggered immunity (PmETI-type) genes, including transcriptional factor (Myc2), pathogenesis-related protein 4 (PR4), and defensin (Def2-2), to block early infection. Additionally, SUMO-protease p0903 is a key regulator of early symbiosis signalling in peanuts (KK5), influencing genes involved in various stages of early signalling such as cyclic nucleotide-gated channels (CNGC), symbiotic remorins (SYMREM), and E3 ubiquitin-protein ligase LIN-1 (CERBERUS). The dotted line indicates the unclear symbiotic mechanism.