While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

The leucine-rich repeat class of receptor-like kinase (LRR-RLKs) encoding genes represents the largest family of putative receptor genes in the Arabidopsis thaliana genome. However, very little is known about the range of biological process that they control. We present in this paper the functional characterization of RLK7 that has all the structural features of a receptor-like kinase of the plant-specific LRR type. To this end, we identified and characterized three independent T-DNA insertion mutants, constructed lines carrying truncated versions of this putative receptor, one lacking the cytoplasmic kinase domain (RLK7Δkin) and the other one lacking 14 LRR repeats (RLK7ΔLRR) and generated RLK7 overexpressing lines. We thus provide evidences that RLK7 is involved in the control of germination speed and the tolerance to oxidant stress. First, consistent with the expression kinetics of the RLK7 gene in the seeds, we found that all three mutants showed a delay in germination, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines displayed a phenotype of more precocious germination. Second, a non-hypothesis driven proteomic approach revealed that in the seedlings of the three T-DNA insertion lines, four enzymes directly or indirectly involved in reactive oxygen species detoxification, were significantly less abundant. Consistent with this finding, the three mutants were less tolerant than the wild type to a hydrogen peroxide treatment, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines presented the opposite phenotype.

Plants are able to reiteratively form new organs in an environmentally adaptive manner during postembryonic development. Organ formation in plants is dependent on stem cell niches (SCNs), which are located in the so-called meristems. Meristems show a functional zonation along the apical-basal axis and the radial axis. Shoot apical meristems of higher plants are dome-like structures, which contain a central SCN that consists of an apical stem cell pool and an underlying organizing center. Organ primordia are formed in the circular peripheral zone (PZ) from stem cell descendants in which differentiation programs are activated. One mechanism to keep this radial symmetry integrated is that the existing SCN actively suppresses stem cell identity in the PZ. However, how this lateral inhibition system works at the molecular level is far from understood. Here, we show that a defect in the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) causes the formation of extra SCNs in the presence of an intact primary shoot apical meristem, which at least partially contributes to the enhanced shoot meristem size and leaf initiation rate found in the mutant. This defect appears to be neither a specific consequence of the altered cytokinin levels in amp1 nor directly mediated by the WUSCHEL/CLAVATA feedback loop. De novo formation of supernumerary stem cell pools was further enhanced in plants mutated in both AMP1 and its paralog LIKE AMP1, indicating that they exhibit partially overlapping roles to suppress SCN respecification in the PZ.

The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure. Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold increase in albicidin production, offering promising strategies for engineering overproduction.Albicidin is produced by Xanthomonas albilineans which is a slow-growing bacterium and the causal agent of sugarcane leaf scald (13). Albicidin is involved in the pathogenicity of X. albilineans and inhibits the replication of chloroplastic DNA (6, 7). Albicidin also inhibits DNA replication in Escherichia coli at nanomolar concentrations (4), whereas mammalian cells are unaffected at 8 g/ml (3). A recent study showed that albicidin targets DNA gyrase with features of inhibition that differ from those of other known antibiotics (11). Albicidin is synthesized by a unique hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway that does not resemble any other pathway described to date. Three genomic regions (XALB1, XALB2, and XALB3) involved in albicidin biosynthesis were cloned and sequenced (12,14,15,17). XALB1 contains the majority of albicidin biosynthetic genes in a cluster of 20 open reading frames (ORFs) (albI to albXX). ORFs albI, albIV, and albIX encode three large PKSs and NRPSs, whereas the other ORFs are resistance, modifying, and regulatory genes. XALB2 and XALB3 each contain a single biosynthetic gene, albXXI and albXXII, whose products are, respectively, a phosphopantetheinyl transferase and the heat shock protein HtpG. The antibiotic activity of albicidin against a wide range of gram-positive and gram-negative pathogenic bacteria (Enterobacter aerogenes, E. coli, Haemophilus influenzae, Klebsiella pneumoniae, Shigella sonnei, and Staphylococcus aureus) is of interest for the development of new antibacterial drugs (4, 5). Low yields of albicidin production in slow-growing X. albilineans have slowed studies of its chemical structure and potential therapeutic applications. A heterologous system for albicidin overproduction is therefore highly desirable (i) to increase the levels of expression of the biosynthetic enzymes, (ii) to obtain large quantities of albicidin, and (iii) to characterize albicidin through nuclear magnetic resonance and mass spectrometry analyses. We report here the construction of a two-plasmid expression system harboring the complete albicidin biosynthetic gene set; its transfer into a fast-growing heterologous host, X. axonopodis pv. vesicatoria; and the subsequent production of albicidin.Two wide-host-range plasmids derived from IncW vector pUFR043 (10) and IncP vector pLAFR3 (16) were used for the transfer of the complete albicidin biosynthetic machinery. The previously described IncW plasmid pALB571 (12, 14) was used to transfer albI to albIX (the insert corresponds to nucleotides 19001 to 55839 of accession no. AJ586576). Plasmid pLAFRK7 was con...

) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homolog HsGCPII, a tumor marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a cytokinin analog. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-related mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a tool to characterize the plant-specific functions of this important class of proteins.Arabidopsis ALTERED MERISTEM PROGRAM1 (AMP1, At3G54720, MEROPS ID: M28.007) belongs to the Zn 2+

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNAdependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem.