Mette Grønlund

Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases

Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes. Despite this host range difference, mycorrhizal and rhizobial invasion shares a common plant-specified genetic programme controlling the early host interaction. One feature distinguishing legumes is their ability to perceive rhizobial-specific signal molecules. We describe here two LysM-type serine/threonine receptor kinase genes, NFR1 and NFR5, enabling the model legume Lotus japonicus to recognize its bacterial microsymbiont Mesorhizobium loti. The extracellular domains of the two transmembrane kinases resemble LysM domains of peptidoglycan- and chitin-binding proteins, suggesting that they may be involved directly in perception of the rhizobial lipochitin-oligosaccharide signal. We show that NFR1 and NFR5 are required for the earliest physiological and cellular responses to this lipochitin-oligosaccharide signal, and demonstrate their role in the mechanism establishing susceptibility of the legume root for bacterial infection.

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.

Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata

Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete beta-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR.

Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum

Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However, the available PEBV-VIGS protocols are inadequate for studying genes involved in the symbiosis with arbuscular mycorrhizal fungi (AMF).Here we describe a PEBV-VIGS protocol suitable for reverse genetics studies in pea of genes involved in the symbiosis with AMF and show its effectiveness in silencing genes involved in the early and late stages of AMF symbiosis.

Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus.

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

Virus-Induced Gene Silencing (VIGS) as a Reverse Genetic Tool to Study Development of Symbiotic Root Nodules

Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the distinct non-nodulating phenotype of nin mutants in P. sativum, Lotus japonicus, and Medicago truncatula. The vector based on Pea early browning virus (PEBV) was engineered to carry one of three nonoverlapping fragments (PsNinA, PsNinB, and PsNinC) derived from the PsNin cDNA. Vector inoculation was mediated by agroinfiltration and, 2 weeks later, a Rhizobium leguminosarum bv. viceae culture was added in order to induce root nodulation. At this time point, it was estimated that systemic silencing was established because leaves of reference plants inoculated with PEBV carrying a fragment of Phytoene desaturase displayed photo bleaching. Three weeks after Rhizobium spp. application, plants inoculated with a control vector nodulated normally, whereas nodulation was almost eliminated in plants inoculated with a vector carrying PsNinA and PsNinC. For plants inoculated with a vector carrying PsNinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired.

Induction of hairy roots for symbiotic gene expression studies

The model legume Lotus japonicus can be transformed and regenerated efficiently with Agrobacterium tumefaciens or A. rhizogenes. However, it takes between 8 to 12 months to obtain seeds of transgenic plants. We therefore developed a rapid and efficient transformation protocol using A. rhizogenes to induce transgenic hairy roots that can be inoculated with Mesorhizobium loti 2 weeks after transformation. The first nodules emerge 8 to 10 days after inoculation, as on the roots of wild type Lotus plants and expression of plant genes involved in any step of nodulation can be completed within two months after the start of a transformation-nodulation experiment. A large number of seedlings can be tranformed in one experiment, allowing addressing of a number of variables in one single tranformationnodulation experiment.

The Lotus japonicus ndxgene family is involved in nodule function and maintenance

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndxgenes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

VECTORS FOR REVERSE GENETICS AND EXPRESSION ANALYSIS

The binary Agrobacterium pPZP211 vector is stable and fully sequenced. We have produced derivatives of this vector that can be used for general over-expression, nodule-specific expression, expression pattern analysis, and protein localization studies.

EMBEDDING ROOT AND NODULE TISSUE IN PLASTIC (BMM)

Immunolocalisation and in situ hybridisation allow the detection of proteins and RNA respectively, in individual cells of different tissue types. Hybridisation on serial 5–8 μm sections is most frequently performed with tissue embedded in paraffin wax but it is often difficult to obtain high-resolution sections from soft tissue with this embedding process. To overcome this, alternative localisation protocols have been developed utilising plastic resins. We have used plastic embedded tissue from Lotus japonicus roots and young nodules successfully in immunolocalisation experiments and developed a protocol that can also be adapted for in situ RNA localisation studies. The different parameters tested are described, as well as the use of alkaline phosphatase- or fluorescently-conjugated secondary antibodies.