The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals

Abstract

SummaryRoot nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation.Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs.We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas.Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

Symbiotic Performance of Diverse Frankia Strains on Salt-Stressed Casuarina glauca and Casuarina equisetifolia Plants

“…To further investigate the effects of salinity during the early stages of the establishment of symbiosis, we studied the impact of salt stress on the expression of two early symbiotic marker genes: CgNIN (Clavijo et al, 2015) and Cg12 (Svistoonoff et al, 2003) using transgenic plants of C. glauca expressing Pro CgNIN:GFP and Pro Cg12:GFP . CgNIN gene is a pre-infection marker which is early expressed in root hairs competent for Frankia infection (Clavijo et al, 2015) and Cg12 , an infection marker associated with root hairs and cortical cells infection by Frankia (Svistoonoff et al, 2003).…”

“…CgNIN gene is a pre-infection marker which is early expressed in root hairs competent for Frankia infection (Clavijo et al, 2015) and Cg12 , an infection marker associated with root hairs and cortical cells infection by Frankia (Svistoonoff et al, 2003). …”

“…The Pro Cg12:GFP line showing the highest expression levels of GFP was clonally propagated as described (Svistoonoff et al, 2010). Similarly for Pro CgNIN:GFP , we used the transgenic line previously described (Clavijo et al, 2015) which showed the highest GFP expression.…”

“…A total of 180 lateral roots and 12,217 root hairs were observed. Root hair deformation intensity was evaluated as described in Clavijo et al (2015). For each micrograph, root hairs were observed and the following scoring was used: 0, no deformation; 1, straight root hair with tip swelling; 2, only one change in growth direction; 3, more than one change in growth direction but no bifurcation; 4, one or more bifurcations.…”

“…Frankia hyphae proceed to penetrate the host plant through a deformed root hair (Franche et al, 1998) and induce the expression of several plant genes involved in actinorhizal nodule formation and functioning. Among them are the CgNIN , encoding a transcriptional factor, expressed at pre-infection stages in root hairs competent for Frankia infection (Clavijo et al, 2015) and Cg12 , encoding a subtilase, whose expression is linked to the infection of root hairs and cortical cells by Frankia (Laplaze et al, 2000; Svistoonoff et al, 2003). …”