An ancient enzyme domain hidden in the putative beta-glucan elicitor receptor of soybean may play an active part in the perception of pathogen-associated molecular patterns during broad host resistance

A successful defense against potential pathogens requires that a host organism is able to discriminate between self and nonself structures. Soybean (Glycine max L.) exploits a specific molecular pattern, a 1,6-beta-linked and 1,3-beta-branched heptaglucoside (HG), present in cell walls of the oomycetal pathogen Phytophthora sojae, as a signal compound eliciting the onset of defense reactions. The specific and high affinity HG-binding site is contained in the beta-glucan-binding protein (GBP), which in turn is part of a proposed receptor complex. The ability to perceive and respond to Phytophthora cell wall-derived beta-glucan elicitors is exclusive to plants that belong to the Fabaceae. However, we propose that the presence of the GBP is essential, but not sufficient for beta-glucan elicitor-dependent disease resistance because genes encoding GBP-related proteins can be retrieved from many plant species. Furthermore, we show that the GBP is composed of two different carbohydrateactive protein domains, one containing the beta-glucan-binding site, and the other related to glucan endoglucosidases of fungal origin. The glucan hydrolase displays most likely an endo-specific mode of action, cleaving only 1,3-beta-d-glucosidic linkages of oligoglucosides consisting of at least four moieties. Thus, the intrinsic endo-1,3-beta-glucanase activity of the GBP is perfectly suited during initial contact with Phytophthora to release oligoglucoside fragments enriched in motifs that constitute ligands for the high affinity binding site present in the same protein. The concept of innate immunity in plants receives substantial support by this highly sophisticated system using ancient enzyme modules as an active part of the recognition mechanism.     

Defensin gene family in <Emphasis Type="Italic">Medicago truncatula:</Emphasis> structure,expression and induction by signal molecules

A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research’s (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.     

Primitive Defence: The MiAMP1 Antimicrobial Peptide Family

The founder of the MiAMP1 protein family was originally isolated from Macadamia integrifolia and had antimicrobial activity in vitro. MiAMP1 was the first plant protein with a structure containing a βγ-crystallin precursor fold, a structural superfamily associated with antimicrobial proteins in other kingdoms. In recent times, expanding plant genomics information has revealed that genes encoding homologues of MiAMP1 are conserved across the plant kingdom from lycophytes, gymnosperms to early angiosperms (e.g. Amborella, Papaver) and some monocots (e.g. Zantedeschia, Zea, Sorghum). Many studies of plant–pathogen interactions in gymnosperms have demonstrated a potential role for MiAMP1 family members in defence against fungal pathogens. This commentary describes the discovery and diversity of this protein family and considers current evidence supporting, and future opportunities for substantiating, a role in defence in primitive plants, and why this role may have diminished in higher plants.     

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high percent (83%) of successful protein identification was obtained when using M. truncatula clustered EST database for queries. During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels. The membrane location of microsomal proteins was further confirmed using PMF identification. This study reports a fractionation process for characterizing microsomal root proteins of M. truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.     

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.     

Genome-wide identification and characterization of putative cytochrome P450 genes in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

In plants, cytochrome P450 is a group of monooxygenases existing as a gene superfamily and plays important roles in metabolizing physiologically important compounds. However, to date only a limited number of P450s have been identified and characterized in legumes. In this study, data mining methods were used, and 151 putative P450 genes in the model legume Medicago truncatula were identified, including 135 novel sequences. These genes were classified into 9 clans and 44 families by sequence similarity, and among those 4 new clans and 21 new families not reported previously in legumes. By comparison of these genes with P450 genes in Arabidopsis and rice, it was found that most of the known P450 families in dicot species exist in M. truncatula. The representative protein sequences of putative P450s were aligned, and the secondary elements were assigned based on the known structure P450BM3. Putative substrate recognition sites (SRSs) and substrate binding sites were also identified in these sequences. In addition, the ESTs-derived expression profiles (digital Northern) of the putative P450 genes were analyzed, which was confirmed by semi-quantitative RT-PCR analyses of several selected P450 genes. These results will provide a base for catalogue information on P450 genes in M. truncatula and for further functional analysis of P450 superfamily genes in legumes.     

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

The divergence and positive selection of the plant‐specific BURP‐containing protein family

BURP domain‐containing proteins belong to a plant‐specific protein family and have diverse roles in plant development and stress responses. However, our understanding about the genetic divergence patterns and evolutionary rates of these proteins remain inadequate. In this study, 15 plant genomes were explored to elucidate the genetic origins, divergence, and functions of these proteins. One hundred and twenty‐five BURP protein‐encoding genes were identified from four main plant lineages, including 13 higher plant species. The absence of BURP family genes in unicellular and multicellular algae suggests that this family (1) appeared when plants shifted from relatively stable aquatic environments to land, where conditions are more variable and stressful, and (2) is critical in the adaptation of plants to adverse environments. Promoter analysis revealed that several responsive elements to plant hormones and external environment stresses are concentrated in the promoter region of BURP protein‐encoding genes. This finding confirms that these genes influence plant stress responses. Several segmentally and tandem‐duplicated gene pairs were identified from eight plant species. Thus, in general, BURP domain‐containing genes have been subject to strong positive selection, even though these genes have conformed to different expansion models in different species. Our study also detected certain critical amino acid sites that may have contributed to functional divergence among groups or subgroups. Unexpectedly, all of the critical amino acid residues of functional divergence and positive selection were exclusively located in the C‐terminal region of the BURP domain. In conclusion, our results contribute novel insights into the genetic divergence patterns and evolutionary rates of BURP proteins.     

Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula

Plant genes induced during early root colonization of Medicago truncatula Gaertn. J5 by a growth-promoting strain of Pseudomonas fluorescens (C7R12) have been identified by suppressive subtractive hybridization. Ten M. truncatula genes, coding proteins associated with a putative signal transduction pathway, showed an early and transient activation during initial interactions between M. truncatula and P. fluorescens, up to 8 d after root inoculation. Gene expression was not significantly enhanced, except for one gene, in P. fluorescens-inoculated roots of a Myc(-)Nod(-) genotype (TRV25) of M. truncatula mutated for the DMI3 (syn. MtSYM13) gene. This gene codes a Ca(2+) and calmodulin-dependent protein kinase, indicating a possible role of calcium in the cellular interactions between M. truncatula and P. fluorescens. When expression of the 10 plant genes was compared in early stages of root colonization by mycorrhizal and rhizobial microsymbionts, Glomus mosseae activated all 10 genes, whereas Sinorhizobium meliloti only activated one and inhibited four others. None of the genes responded to inoculation by either microsymbiont in roots of the TRV25 mutant. The similar response of the M. truncatula genes to P. fluorescens and G. mosseae points to common molecular pathways in the perception of the microbial signals by plant roots.