Summary Dalbergoids are typified by crack‐entry symbiosis which is evidenced to be Nod Factor (NF)‐independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF‐producing, prompting us to check whether symbiosis in this legume could also be NF‐independent. For this, we followed the symbiosis with two NF‐containing bradyrhizobial strains – SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF‐deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis‐Oryza intercropped field. While SEMIA6144ΔnodC was non‐nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF‐deficient strains – BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF‐independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF‐dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF‐dependent legume, Arachis does retain a vestigial, less‐efficient form of NF‐independent symbiosis.

No abstract

Rhizobia-legume interactions recruit cytokinin for the induction of nodule primordia in the cortex. Cytokinin signaling regulates auxin transport and biosynthesis, causing local auxin accumulation which triggers cortical cell division. Since sugar signaling can trigger auxin responses, we explored whether sugar treatments could rescue symbiosis in the Medicago truncatula Cytokinin Response 1 mutant (cre1.) Herein we demonstrate that sucrose and its nonmetabolizable isomer turanose can trigger auxin response and recover functional symbiosis in cre1, indicating sucrose signaling to be necessary for restoration of symbiosis. In both M. truncatula A17 (wild-type) and cre1, sucrose signaling significantly upregulated IAA-Ala Resistant 3 (IAR33), encoding an auxin conjugate hydrolase, in rhizobia-infected as well as in uninfected roots. Knockdown of IAR33 (IAR33-KD) significantly reduced nodulation in A17, highlighting the importance of deconjugation-mediated auxin accumulation during nodule inception. In cre1, IAR33-KD restricted the sucrose mediated restoration of functional symbiosis, suggesting deconjugation-mediated auxin accumulation plays a key role in the absence of CRE1-mediated auxin biosynthesis and transport control. Overexpression of IAR33 also restored functional symbiosis in cre1, further suggesting thatIAR33 mediates auxin accumulation in response to sucrose signaling. Since all the observed sucrose mediated responses were common to A17 and cre1, deconjugation-mediated auxin response appeared to be independent of CRE1, whichnormally governs local auxin accumulation in the presence of rhizobia. We propose that sucrose-dependent restoration of symbiosis in cre1 occurs by the activation of IAR33-mediated auxin deconjugation.

Rhizobia-legume interaction recruits cytokinin for the induction of nodule primordia in the cortex. In Medicago truncatula, cytokinin signalling involves flavonoid mediated local alteration of polar auxin transport for triggering cortical cell division. Since sugar signalling is widely evidenced to trigger auxin responses, we explored whether sugar treatment could compensate for cytokinin signalling in M. truncatula cytokinin perception mutant cre1. Herein we demonstrate that turanose, a non-metabolizable sucrose analogue can trigger auxin response and show signs of recovery of symbiosis in cre1. Additionally, turanose upregulated the expression of WUSCHEL-related homeobox 5 (MtWOX5) which prompted us to check if overexpression of WOX5 could rescue cre1. Intriguingly, while overexpression of MtWOX5 failed, WOX5 from Arachis hypogaea (AhWOX5) completely restored functional symbiosis in cre1 with an efficiency resembling the wildtype. This indicate that indeterminate and determinate WOX5 responds to differential cues for nodule development and is consistent with their distinct clustering in distance trees. The mechanism of compensation of cytokinin signalling for recovery of symbiosis in cre1 was distinct for turanose treatment and AhWOX5 expression. Turanose increased MtHK2 level by ∼5-fold and doubled the level of MtRR4 whereas AhWOX5 elevated the level of MtRR4 by ∼12-fold without affecting the level of MtHK2/3. Thus, turanose compensated MtCRE1 at receptor level whereas WOX5 restored the signalling at the level of response regulators. We propose a working model to discuss how sugar mediated WOX5 signalling have compensated MtCRE1 to recover normal progress of symbiosis in cre1.One sentence summarySugar signalling rescues symbiosis in Medicago truncatula cytokinin perception mutant cre1

Nitrogenase that catalyses anaerobic symbiotic nitrogen fixation (SNF) in legumes is synthesized by rhizobium. Legume root cells express nodulin proteins after infection with rhizobia. Nodulins have been classified as early and late, reflecting the time points of their expression. Leghemoglobin (LegH), which is a classic example of a late nodulin, sequesters oxygen inside the nodule to protect the nitrogenase from oxygen toxicity to sustain SNF. Previous data from our laboratory demonstrated that phosphorylated LegH at S45 showed compromised oxygen sequestration in vitro due to structural disruption of the porphyrin binding pocket responsible for its oxygen binding. Moreover, we have demonstrated by using co-immunoprecipitation that LegH interacts both in vitro with Nodulin 16 of Lotus japonicus (Nlj16), another late nodulin. Fluorescence Immunohistochemistry (IHC) data shows that both LegH and Nlj16 are localized in the membrane and cytosol of infected cells. Notably, serine phosphorylation of LegH and interaction of Nlj16 with LegH respectively reduces and increases its in vitro oxygen sequestration ability. In this report, to further elucidate the spatio-temporal regulation of this interaction, we generated hairy root transgenic Lotus japonicus plants where Nlj16 has been knocked down by using siRNA. Most interestingly, our data shows that the membrane localization of LegH is obliterated in the Nlj16 knocked-down root cells infected with the cognate Rhizobia suggesting a distinct role of Nlj16 in its recruitment to the membrane of nodule cells.