Appressorial Melanization Research Articles

Deficiency of ChPks and ChThr1 Inhibited DHN-Melanin Biosynthesis, Disrupted Cell Wall Integrity and Attenuated Pathogenicity in Colletotrichum higginsianum.

Colletotrichum higginsianum is a major pathogen causing anthracnose in Chinese flowering cabbage (Brassica parachinensis), posing a significant threat to the Chinese flowering cabbage industry. The conidia of C. higginsianum germinate and form melanized infection structures called appressoria, which enable penetration of the host plant's epidermal cells. However, the molecular mechanism underlying melanin biosynthesis in C. higginsianum remains poorly understood. In this study, we identified two enzymes related to DHN-melanin biosynthesis in C. higginsianum: ChPks and ChThr1. Our results demonstrate that the expression levels of genes ChPKS and ChTHR1 were significantly up-regulated during hyphal and appressorial melanization processes. Furthermore, knockout of the gene ChPKS resulted in a blocked DHN-melanin biosynthetic pathway in hyphae and appressoria, leading to increased sensitivity of the ChpksΔ mutant to cell-wall-interfering agents as well as decreased turgor pressure and pathogenicity. It should be noted that although the Chthr1Δ mutant still exhibited melanin accumulation in colonies and appressoria, its sensitivity to cell-wall-interfering agents and turgor pressure decreased compared to wild-type strains; however, complete loss of pathogenicity was not observed. In conclusion, our results indicate that DHN-melanin plays an essential role in both pathogenicity and cell wall integrity in C. higginsianum. Specifically, ChPks is crucial for DHN-melanin biosynthesis while deficiency of ChThr1 does not completely blocked melanin production.

Non-host resistance of arabidopsis and apple is associated with callose accumulation and changes in preinfective structures of Colletotrichum species

Non-host resistance (NHR) describes the immunity state of plant species against non-adapted pathogen species and has been considered the most durable and effective form of plant resistance in nature. The objective of this work was to study the non-host resistance mechanisms involved in Arabidopsis thaliana and Malus domestica against Colletotrichum fructicola and Colletotrichum higginsianum, respectively. For this, the development of preinfective structures of Colletotrichum species, hypersensitivity responses and callose accumulation were monitored in host and non-host plants. Conidial germination and appressorial formation of Colletotrichum were affected on non-host leaf surfaces. Conidial germination and appressorial melanization of both Colletotrichum species occurred in a faster manner on arabidopsis than on apple leaves. On apple leaves, appressoria of C. higginsianum became more pedicelate, while on its host (arabidopsis) they were typically sessile. Hypersensitive response (HR) and appressorium-associated HR occurred at low frequency in epidermal cells of both plants, and no relationship could be established with NHR. Callose accumulation was significantly higher in inoculated non-host plants, mainly at attempted entry sites of Colletotrichum. Our results demonstrate that the mechanisms of NHR involved in this heterologous interaction were associated to pre-invasive events as demonstrated by the changes in development of preinfective structures and the accumulation of papillary callose at sites of penetration attempts.

LAC2 Encoding a Secreted Laccase Is Involved in Appressorial Melanization and Conidial Pigmentation in Colletotrichum orbiculare

Both Colletotrichum and Magnaporthe spp. develop appressoria pigmented with melanin, which is essential for fungal pathogenicity. 1,8-Dihydroxynaphthalene (1,8-DHN) is believed to be polymerized to yield melanin around the appresorial cell wall through the oxidative activity of laccases. However, no 1,8-DHN laccase has yet been identified in either Colletotrichum or Magnaporthe spp. Here, we report a laccase gene, LAC2, that is involved in the appressorial melanization of Colletotrichum orbiculare, which causes cucumber anthracnose. LAC2 encodes a protein with a signal peptide and has high homology to fungal laccases. The conidial color of lac2 mutants is distinct from that of the C. orbiculare wild type, and the mutants are nonpathogenic. Notably, the mutant appressoria are defective in melanization, and a host invasion assay showed that the appressoria are nonfunctional. LAC2 was induced during appressorial melanization. These results suggest that LAC2 oxidizes 1,8-DHN in the appressoria. The LAC2 homologues of other fungi located in the same phylogenetic clade as LAC2 fully complemented the lac2 mutants. Interestingly, a LAC2 homologue, located in a different clade, complemented the conidial pigmentation but not appressorial melanization of the mutants, suggesting that the LAC2 function in appressorial melanization might only be conserved in laccases of the LAC2 clade.

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.

Discovery of Pathogenicity Genes in the Crucifer Anthracnose Fungus Colletotrichum higginsianum, Using Random Insertional Mutagenesis

Agrobacterium tumefaciens-mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-beta2, ornithine decarboxylase, beta-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.

Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium.

In Colletotrichum lagenarium, which is the causal agent of cucumber anthracnose, PEX6 is required for peroxisome biogenesis and appressorium-mediated infection. To verify the roles of peroxisome-associated metabolism in fungal pathogenicity, we isolated and functionally characterized ICL1 of C. lagenarium, which encodes isocitrate lyase involved in the glyoxylate cycle in peroxisomes. The icl1 mutants failed to utilize fatty acids and acetate for growth. Although Icl1 has no typical peroxisomal targeting signals, expression analysis of the GFP-Icl1 fusion protein indicated that Icl1 localizes in peroxisomes. These results indicate that the glyoxylate cycle that occurs inside the peroxisome is required for fatty acid and acetate metabolism for growth. Importantly, in contrast with the pex6 mutants that form nonmelanized appressoria, the icl1 mutants formed appressoria that were highly pigmented with melanin, suggesting that the glyoxylate cycle is not essential for melanin biosynthesis in appressoria. However, the icl1 mutants exhibited a severe reduction in virulence. Appressoria of the icl1 mutants failed to develop penetration hyphae in the host plant, suggesting that ICL1 is involved in host invasion. The addition of glucose partially restored virulence of the icl1 mutant. Heat shock treatment of the host plant also enabled the icl1 mutants to develop lesions, implying that the infection defect of the icl1 mutant is associated with plant defense. Together with the requirement of PEX6 for appressorial melanization, our findings suggest that peroxisomal metabolic pathways play functional roles in appressorial melanization and subsequent host invasion steps, and the latter step requires the glyoxylate cycle.

Epicatechin and catechin may prevent coffee berry disease by inhibition of appressorial melanization of Colletotrichum kahawae

Colletotrichum kahawae is the causal agent of coffee berry disease. Appressorial melanization is essential for the fungal penetration of plant cuticle. Epicatechin is abundant in green coffee berry pericarp. Inoculation of C. kahawae conidial suspension containing 1.2 mg epicatechin or catechin/ml did not affect conidial germination or appressorial formation but appressorial melanization was completely inhibited and infection by the treated conidia was less than 30% of the untreated control. Epicatechin and catechin may, therefore, prevent coffee berry disease by inhibition of the appressorial melanization of C. kahawae.

Colletotrichum: tales of forcible entry, stealth, transient confinement and breakout

Summary Taxonomy: Imperfect, anamorphic fungus (subdivision Deuteromycotina, form-class Deuteromycetes, form-subclass Coelomycetidae, form-order Melanconiales, form-family Melanconiaceae) with 39 'accepted' species [Sutton, B.C. (1992) The genus Glomerella and its anamorph Colletotrichum. In: Colletotrichum: Biology, Pathology and Control (Bailey, J.A. and Leger, M.J., eds). Wallingford, UK: CAB International, pp. 1-26.] which continue to be revised and clarified by molecular taxonomic techniques. Species complexes and subspecific groups have been proposed. Species of Colletotrichum attack a large number of important tropical and sub-tropical crop species and cause economically significant diseases of cereals, grain legumes, vegetables, forage legumes, fruit crops and perennial crops. Tropical and sub-tropical fruit production is significantly affected by postharvest anthracnose. Disease symptoms: Symptoms of the attack are commonly known as anthracnose and comprise dark, sunken, lenticular necrotic lesions containing the acervuli of the pathogen. Key attractions: A model fungus for research on host specificity, mycoherbicides, appressorial melanization, appressorial function, quiescent infection, fungal lifestyles, intracellular hemibiotrophy and the determinants of the switch from biotrophy to necrotrophy among others. http://www.uark.edu/depts/plant/, http://www.sorghumanthracnose.org/, http://www.iacr.bbscr.ac.uk/ppi/staff/roc_rc.html.

Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium and Pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 binuclear cluster DNA‐binding motifs and regulate transcription of melanin biosynthesis genes in a developmentally specific manner

Colletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichummelanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA‐binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1Δ mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Target Site of a New Antifungal Compound KC10017 in the Melanin Biosynthesis ofMagnaporthe grisea

A new experimental fungicide KC10017 (3-[4′-bromo-2′, 6′-dimethylphenoxy]methyl-4-[(3″-methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one) completely controlled rice blast disease at more than 1.0 μg/ml without curative activity. When the chemical was applied after the inoculation was made with injury, the fungicide had no effect on controlling rice blast. The fungicide at concentrations of more than 1 μg/ml blocked appressorial melanization and penetration of cellophane membrane byMagnaporthe griseaP-2 without affecting fungal growth, spore germination, and appressorial formation. To elucidate the target site of KC10017 in melanin biosynthesis inhibition,M. griseaP-2 was grown in potato dextrose broths treated with both KC10017 and tricyclazole. The mycelia and culture media of cultures treated with both KC10017 and tricyclazole at a concentration of 5 μg/ml each became colorless like as the cultures treated with KC10017 alone, whereas the tricyclazole-treated cultures exhibited red color. Shunt products of 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,6,8-trihydroxy-1-(2H)-naphthalenone, and 4-hydroxyscytalone were absent or present in only trace amounts in the cultures treated with KC10017 alone or both KC10017 and tricyclazole. However, they were detected at much higher levels in the cultures treated with tricyclazole alone. On the other hand, appressorial melanization and penetration ability inM. griseatreated with KC10017 was recovered by the addition of scytalone. These results demonstrate that the target site of KC10017 in melanin biosynthesis inhibition is at the pentaketide synthesis step and/or at the pentaketide cyclization step prior to 1,3,6,8-tetrahydroxynaphthalene formation.

Exogenous scytalone restores appressorial melanization and pathogenicity in albino mutants ofColletotrichum graminicola

(1989). Exogenous scytalone restores appressorial melanization and pathogenicity in albino mutants of Colletotrichum graminicola. Canadian Journal of Plant Pathology: Vol. 11, No. 4, pp. 349-352.

Effect of pentachloronitrobenzene, pentachloroaniline, and albinism on epidermal penetration by appressoria of Pyricularia

Pentachloroaniline or pentachloronitrobenzene at concentrations between 0.5 and 5 μg/ml blocked appressorial melanization and appressorial penetration of onion epidermal cell walls by Pyricularia oryzae, strain P-2, without affecting conidial germination or appressorial formation. Among polyphenol oxidase inhibitors, salicylhydroxamic acid, and phenylthiourea at 100 μg/ml inhibited appressorial penetration of onion epidermis without affecting appressorial melanization, whereas sodium diethyldithiocarbamate and 2,3-naphthalenediol did not affect either process at 1.0 μg/ml but inhibited conidial germination and appressorial formation at 10 μg/ml. EDTA was without effect on either melanization or penetration at 100 μg/ml. The melanized appressoria of Pyricularia grisea, strain BL-3, penetrated onion epidermal cell walls, whereas nonpigmented appressoria of an albino mutant (AL-3) of this fungus did not. Melanization and penetration ability of AL-3 appressoria were restored by adding melanin biosynthesis intermediates scytalone or 1,8-dihydroxynaphthalene (1,8-DHN). Tricyclazole blocked melanization and epidermal penetration by AL-3 appressoria treated with scytalone but not by those treated with 1,8-DHN. Scytalone (20 μg/ml) increased the effectiveness of tricyclazole in blocking penetration by appressoria of BL-3. Tricyclazole was about 10 times more effective in inhibiting penetration by P-2 appressoria than by BL-3 appressoria.

Effect of inhibitors of melanin biosynthesis on structure and function of appressoria of Colletotrichum lindemuthianum

An investigation was made of the mechanism by which the compounds 4,5-dihydro-4-methyltetrazolo[1,5- a]quinazolin-5-one (pp389); tricyclazole; and pyroquilon protect Phaseolus vulgaris from infection by the fungus Colletotrichum lindemuthianum. These compounds prevented infection of unwounded plant tissue, but not of tissue with a punctured epidermis. The compounds were not toxic to the pathogen at concentrations which blocked appressorial melanization and penetration of epidermal cell walls of bean and Bryophyllum. The compounds greatly increased the frequency of appressoria that produced non-penetrating hyphae which grew laterally along the epidermal surface. Control appressoria (52%) penetrated Formvar (polyvinyl formal) plastic membranes 100–140 nm thick, while only 2% of those treated with 10 μg ml −1 tricyclazole penetrated the membranes. The walls of untreated appressoria contained an electron-dense layer that was absent from walls of appressoria treated with pp389. The electron density of this layer increased in the region surrounding the infection pore. The basal part of untreated appressoria was conical in shape and focused on a relatively small area of the cuticle. Treated appressoria were poorly focused with a large flat basal area in contact with the cuticle. This study suggests that the unmelanized walls of treated appressoria lack the rigidity necessary to support the mechanical forces required for penetrating the plant cuticle.

Action of the antipenetrant, tricyclazole, on appressoria of Pyricularia oryzae

The antipenetrant, tricyclazole, blocked melanin biosynthesis and protected unwounded, but not wounded, rice plants from infection by a wild-type isolate of Pyricularia oryzae. Two buff mutants genetically deficient in melanin biosynthesis infected wounded but not unwounded plants. Tricyclazole (0·1–10 μg ml−1) did not inhibit spore germination or appressorial formation by spores of P. oryzae on detached epidermal sections from Bryophyllum pinnatum, but completely blocked appressorial melanization and prevented fungal penetration of the epidermal cell walls. The walls were penetrated by 80–95% of melanized control appressoria. Penetration of 135–145 nm Formvar (polyvinyl formal) plastic membranes was accomplished by 54–77% of control appressoria but only by 0–2% of tricyclazole treated appressoria. The cutinase inhibitor, diisopropylfluorophosphate, at 50 μm or less had little or no effect on spore germination or penetration of B. pinnatum epidermal walls. Concentrations of 100 and 150 μm strongly inhibited penetration, but were also toxic to fungal growth. Melanized walls of control appressoria were impermeable to sucrose and various other solutes, but walls of tricyclazole treated appressoria were freely permeable to these solutes. Ultra-structural studies revealed an electron-dense layer in walls of control appressoria which thickened at the base to form a reinforced region surrounding the periphery of the thin underwall in contact with the cuticle. This electron-dense layer was absent in walls of treated appressoria. The data suggest that failure of tricyclazole treated appressoria to accomplish cuticular penetration is due to the lack of rigidity of the unmelanized appressorial walls.