A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules

Abstract

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus , for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia interactions.

VC1 catalyzes a key step in the biosynthesis of vicine from GTP in faba bean

“…The sterilized seeds were germinated on petri dishes lined with moist filter paper and transferred to magenta boxes containing moist vermiculite. Plants were grown at 21 °C with a photoperiod of 16/8 h. In parallel, plasmids LjUbi:YFP 16 and LjUbi:VC1 were conjugated into Agrobacterium rhizogenes GV3101 using triparental mating 17 . We then infected the in-vitro- grown plants with the transformed A. rhizogenes using a protocol adapted from Kereszt et al 18 .…”

No Home without Hormones: How Plant Hormones Control Legume Nodule Organogenesis

“…Gravitropic stimulation of roots, which results in asymmetric auxin distribution, is also sufficient to alter nodule positioning in L . japonicus ( Nadzieja et al., 2019 ), and application of auxin import inhibitors before rhizobia infection reduced the number of nodules in M . truncatula ( Roy et al., 2017 ).…”

“…japonicus and M . truncatula demonstrated that auxin signaling starts to emerge in cell layers underneath infected root hairs and is strongest in the cortex and pericycle of young nodules ( Pacios-Bras et al., 2003 ; Suzaki et al., 2012 ; Breakspear et al., 2014 ) and in the vasculature and apex of mature nodules ( Takanashi et al., 2011 ; Suzaki et al., 2012 ; Guan et al., 2013 ; Breakspear et al., 2014 ; Franssen et al., 2015 ; Nadzieja et al., 2019 ). These observations support a requirement for auxin signaling in both infection and nodule organogenesis and may relate to the requirement for cell-cycle initiation and progression that occurs in both these processes.…”

“…Although some ethylene responsive markers have been described, none so far developed offers the high-resolution and dynamic range available for CK and auxin (DR5/DII-VENUS reporter; Nadzieja et al., 2019 ), making it challenging to identify the precise location and signaling of ethylene responses during nodule organogenesis. Novel approaches such as single-cell sequencing might circumvent this restriction or lead to the development of such ethylene markers in the future.…”

Natural variation identifies aPxygene controlling root vascular organization and formation of nodules and lateral roots inLotus japonicus

“…Additional transcription factors (NSP1, NSP2, ERN1 and NIN) are then required for infection thread formation [23][24][25][26][27][28][29] involving a pectate lyase encoded by Npl, actin rearrangement by NAP1, PIR1, SCARN, ARPC1 and DREPP, an E3 ubiquitin ligase encoded by Cerberus, an atypical receptor kinase encoded by RinRK, a subunit of mediator complex encoded by Lan, and novel functions encoded by Rpg, Vapyrin and CBS [30][31][32][33][34][35][36][37][38][39][40] . The simultaneous development of nodule organs is mediated by NSP1, NSP2, ERN1, NIN and NF-Ys transcription factors 41,42 that together with localized changes in plant hormone homeostasis (cytokinin, auxin and jasmonic acid) regulate initiation of infection thread formation and cell divisions leading to nodule organogenesis [43][44][45][46] . In contrast, ethylene and abscisic acid suppress nodule formation [47][48][49] .…”