Faculty of Natural Sciences

TY - JOUR

T1 - A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules

AU - Nadzieja, Marcin

AU - Stougaard, Jens

AU - Reid, Dugald

PY - 2019/8

Y1 - 2019/8

N2 - 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.

AB - 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.

KW - auxin

KW - cytokinin

KW - nodulation and N fixation 2

KW - lotus japonicus

KW - symbiosis

KW - BOX PROTEIN TIR1

KW - LOTUS-JAPONICUS

KW - AUXIN RESPONSE

KW - IN-VIVO

KW - CYTOKININ

KW - GENES

KW - PERCEPTION

KW - INHIBITION

KW - EXPRESSION

KW - RHIZOBIUM

U2 - 10.3389/fpls.2019.01000

DO - 10.3389/fpls.2019.01000

M3 - Journal article

C2 - 31428118

VL - 10

JO - Frontiers in Plant Science

JF - Frontiers in Plant Science

SN - 1664-462X

M1 - 1000

ER -