Plant-Microbe Symbiosis

Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) ofGmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.

The interaction between legumes and arbuscular mycorrhizal (AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like (LjMAMI) transcription factor (TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate (Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of LjMAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive LjMAMI in non-symbiotic functions: i.e. root growth.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

To date, little research has been published that quantifies the impact of soil desiccation (e.g. drought) on endemic B. japonicum populations. Pena-Cabriales and Alexander (1979) reported a biphasic decline inRhizobium japonicum numbers in soils undergoing drying. The initial rapid phase of R. japonicum loss coincided with major water loss (simulated drought) whereas the secondary and subsequently slower decline in numbers was governed by the soil water content of specific soils. Pena-Cabriales and Alexander (1979) also noted that organic matter content provided little protection againstR. japonicum desiccation and loss. In a parallel study Oso-Afiana and Alexander (1982) reported similar results when comparing strains of R. japonicum and cowpea rhizobia under desiccation. As one would expect, variation exists among Rhizobium spp. as well as individual isolates within species in their response to soil desiccation, though significant losses were evident regardless of species or isolate (Foulds, 1971; Trotman and Weaver, 1995).