The N-Glycan cluster from Xanthomonas campestris pv. campestris: a toolbox for sequential plant N-glycan processing.

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the ß-xylosidase NixI (GH3), which is involved in the cleavage of the ß-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.

The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.

UNLABELLED: N-Acetylglucosamine (GlcNAc), the main component of chitin and a major constituent of bacterial peptidoglycan, is present only in trace amounts in plants, in contrast to the huge amount of various sugars that compose the polysaccharides of the plant cell wall. Thus, GlcNAc has not previously been considered a substrate exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, expresses a carbohydrate utilization system devoted to GlcNAc exploitation. In addition to genes involved in GlcNAc catabolism, this system codes for four TonB-dependent outer membrane transporters (TBDTs) and eight glycoside hydrolases. Expression of all these genes is under the control of GlcNAc. In vitro experiments showed that X. campestris pv. campestris exploits chitooligosaccharides, and there is indirect evidence that during the early stationary phase, X. campestris pv. campestris recycles bacterium-derived peptidoglycan/muropeptides. Results obtained also suggest that during plant infection and during growth in cabbage xylem sap, X. campestris pv. campestris encounters and metabolizes plant-derived GlcNAc-containing molecules. Specific TBDTs seem to be preferentially involved in the consumption of all these plant-, fungus- and bacterium-derived GlcNAc-containing molecules. This is the first evidence of GlcNAc consumption during infection by a phytopathogenic bacterium. Interestingly, N-glycans from plant N-glycosylated proteins are proposed to be substrates for glycoside hydrolases belonging to the X. campestris pv. campestris GlcNAc exploitation system. This observation extends the range of sources of GlcNAc metabolized by phytopathogenic bacteria during their life cycle. IMPORTANCE: Despite the central role of N-acetylglucosamine (GlcNAc) in nature, there is no evidence that phytopathogenic bacteria metabolize this compound during plant infection. Results obtained here suggest that Xanthomonas campestris pv. campestris, the causal agent of black rot disease on Brassica, encounters and metabolizes GlcNAc in planta and in vitro. Active and specific outer membrane transporters belonging to the TonB-dependent transporters family are proposed to import GlcNAc-containing complex molecules from the host, from the bacterium, and/or from the environment, and bacterial glycoside hydrolases induced by GlcNAc participate in their degradation. Our results extend the range of sources of GlcNAc metabolized by this phytopathogenic bacterium during its life cycle to include chitooligosaccharides that could originate from fungi or insects present in the plant environment, muropeptides leached during peptidoglycan recycling and bacterial lysis, and N-glycans from plant N-glycosylated proteins present in the plant cell wall as well as in xylem sap.

The conserved PFT1 tandem repeat is crucial for proper flowering in Arabidopsis thaliana.

It is widely appreciated that short tandem repeat (STR) variation underlies substantial phenotypic variation in organisms. Some propose that the high mutation rates of STRs in functional genomic regions facilitate evolutionary adaptation. Despite their high mutation rate, some STRs show little to no variation in populations. One such STR occurs in the Arabidopsis thaliana gene PFT1 (MED25), where it encodes an interrupted polyglutamine tract. Although the PFT1 STR is large (∼270 bp), and thus expected to be extremely variable, it shows only minuscule variation across A. thaliana strains. We hypothesized that the PFT1 STR is under selective constraint, due to previously undescribed roles in PFT1 function. We investigated this hypothesis using plants expressing transgenic PFT1 constructs with either an endogenous STR or synthetic STRs of varying length. Transgenic plants carrying the endogenous PFT1 STR generally performed best in complementing a pft1 null mutant across adult PFT1-dependent traits. In stark contrast, transgenic plants carrying a PFT1 transgene lacking the STR phenocopied a pft1 loss-of-function mutant for flowering time phenotypes and were generally hypomorphic for other traits, establishing the functional importance of this domain. Transgenic plants carrying various synthetic constructs occupied the phenotypic space between wild-type and pft1 loss-of-function mutants. By varying PFT1 STR length, we discovered that PFT1 can act as either an activator or repressor of flowering in a photoperiod-dependent manner. We conclude that the PFT1 STR is constrained to its approximate wild-type length by its various functional requirements. Our study implies that there is strong selection on STRs not only to generate allelic diversity, but also to maintain certain lengths pursuant to optimal molecular function.

Cell autonomous and non-cell autonomous control of rhizobial and mycorrhizal infection in Medicago truncatula.

Legumes can form a nitrogen fixing symbiosis with soil bacteria called rhizobia (the RL symbiosis). They can also, like most plants, form symbiotic associations with arbuscular mycorrhizal (AM) fungi, which facilitate plants' phosphate nutrition. In both interactions, the symbionts are hosted inside the plant root. Nitrogen-fixing rhizobia are housed in intracellular symbiotic structures within nodules, while AM fungi form intracellular symbiotic structures, called arbuscules, within cortical root cells. These two endosymbioses present other similarities, including production by the microsymbionts of lipo-chitooligosaccharidic signals (Nod Factors and Myc-LCOs), and the involvement of common plant signaling elements. In Medicago truncatula, DMI3 encodes a calcium and calmodulin dependent protein kinase that is part of this common signaling pathway, while NFP encodes a LysM domain receptor-like kinase involved in Nod Factor perception. Using tissue specific promoters, we recently uncoupled the roles of NFP and DMI3 in the cortex and the epidermis of the root during the RL symbiosis. (1) Here, we provide additional data showing a cell autonomous tissular contribution of DMI3 in the AM symbiosis, and we comment on a non-cell autonomous cortical role of NFP during rhizobial infection.

Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula.

Legumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium- and calmodulin-dependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.