Fig. 3: Sup3, a putative SUMO-protease conferring the ability to nodulate A. indica.

Images of roots and nodules of A. indica plants at 21 days post inoculation with strain NAS96.2 and its mutant derivatives in T3SS (ΩrhcN) and in different genes encoding putative SUMO-proteases (Ωsup1, Ωsup2 and Ωsup3) (A) and strain WSM1744 containing a sup3 homolog and its mutant derivatives (ΩrhcN, Ωsup3, ∆sup3 and ∆sup3::sup3) (B) (scale bars: upper panels 0.5 cm, lower panels 0.2 cm). Nodule number of A. indica plants at 21 days after inoculation with NAS96.2 (C) or WSM1744 (D) and their respective mutants. Box plots show the results of one of the two experiments performed independently (8 plants each). **p ≤ 0.001, significant differences between the wild-type strain NAS96.2 and each mutant (C) and **p ≤ 0.005, ***p < 0.0005, ****p < 0.0001, significant differences between the wild-type strain WSM1744 and each mutant (D) using a nonparametric Kruskal–Wallis test, NS not significant. E Phylogenetic tree of full-length SUMO-proteases identified in the collection of Bradyrhizobium strains used in this study. Considering the very different branch lengths, the MAD (minimal ancestor deviation) method was used for rooting the tree [59]. The SUMO-proteases Sup3 from NAS96.2 and Bel2-5 from USDA61 (indicated by black triangles) belong to two distinct phylogroups (indicated by a color strip). The symbiotic phenotype of the strains in which the SUMO-protease are identified is indicated by a colored range referenced in the key and the presence of an ernA homolog by a red circle. As references (white range), the following SUMO-proteases are included: (i) mlr6316 identified in Mesorhizobium loti MAFF303099 [60]; (ii) SFHH103_04358 identified in Sinorhizobium fredii HH103 [61] and (iii) The XopD SUMO-proteases [57] identified in various Xanthomonas campestris strains.