Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

Experimental evaluation of biological regeneration of arable soil: The effects of grass-clover leys and arbuscular mycorrhizal inoculants on wheat growth, yield, and shoot pathology.

Wheat yields have plateaued in the UK over the last 25 years, during which time most arable land has been annually cropped continuously with short rotations dominated by cereals. Arable intensification has depleted soil organic matter and biology, including mycorrhizas, which are affected by tillage, herbicides, and crop genotype. Here, we test whether winter wheat yields, mycorrhization, and shoot health can be improved simply by adopting less intensive tillage and adding commercial mycorrhizal inoculum to long-term arable fields, or if 3-year grass-clover leys followed direct drilling is more effective for biological regeneration of soil with reduced N fertiliser. We report a trial of mycorrhization, ear pathology, and yield performance of the parents and four double haploid lines from the Avalon x Cadenza winter wheat population in a long-term arable field that is divided into replicated treatment plots. These plots comprised wheat lines grown using ploughing or disc cultivation for 3 years, half of which received annual additions of commercial arbuscular mycorrhizal (AM) inoculum, compared to 3-year mown grass-clover ley plots treated with glyphosate and direct-drilled. All plots annually received 35 kg of N ha-1 fertiliser without fungicides. The wheat lines did not differ in mycorrhization, which averaged only 34% and 40% of root length colonised (RLC) in the ploughed and disc-cultivated plots, respectively, and decreased with inoculation. In the ley, RLC increased to 52%. Two wheat lines were very susceptible to a sooty ear mould, which was lowest in the ley, and highest with disc cultivation. AM inoculation reduced ear infections by >50% in the susceptible lines. In the ley, yields ranged from 7.2 to 8.3 t ha-1, achieving 92 to 106% of UK average wheat yield in 2018 (7.8 t ha-1) but using only 25% of average N fertiliser. Yields with ploughing and disc cultivation averaged only 3.9 and 3.4 t ha-1, respectively, with AM inoculum reducing yields from 4.3 to 3.5 t ha-1 in ploughed plots, with no effect of disc cultivation. The findings reveal multiple benefits of reintegrating legume-rich leys into arable rotations as part of a strategy to regenerate soil quality and wheat crop health, reduce dependence on nitrogen fertilisers, enhance mycorrhization, and achieve good yields.

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors.

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans.

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the alpha1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the alpha1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1delta null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence.

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence.

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth.

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.

Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene.

Conidia (spores) of Colletotrichum lindemuthianum, a fungal plant pathogen causing bean anthracnose, adhere to the aerial parts of host plants to initiate the infection process. These spores possess a fibrillar 'spore coat' as well as a cell wall. In a previous study a mAb, UB20, was raised that recognized glycoproteins on the spore surface. In this study UB20 was used to localize and characterize these glycoproteins and to investigate their possible role in adhesion. Glycoproteins recognized by UB20 were concentrated on the outer surface of the spore coat and, to a lesser extent, at the plasma membrane/cell wall interface. Extraction of spores with hot water or 0.2% SDS resulted in removal of the spore coat. Western blotting with UB20 showed that a relatively small number of glycoproteins were extracted by these procedures, including a major component at 110 kDa. Biotinylation of carbohydrate moieties, together with cell fractionation, confirmed that these glycoproteins were exposed at the surface of the spores. In adhesion assays, > 90% of ungerminated conidia attached to polystyrene Petri dishes within 30 min. UB20 IgG at low concentrations inhibited attachment in an antigen-specific manner. This suggests that the glycoproteins recognized by this mAb may function in the initial rapid attachment of conidia to hydrophobic substrata. Polystyrene microspheres bound selectively to the 110 kDa glycoprotein in Western blots, providing further evidence that this component could mediate interactions with hydrophobic substrata.