Colletotrichum gloeosporioides, an important phytopathogenic fungus, mainly infects tropical fruits and results in serious anthracnose. Previous studies have shown that melanin biosynthesis inhibitor can inhibit the melanization of the appressoria of Magnaporthe grisea and Colletotrichum orbiculare, resulting in limited infection of the hosts. In this study, we identified and characterized a scytalone dehydratase gene (CgSCD1) from C. gloeosporioides which is involved in melanin synthesis. The CgSCD1 gene deletion mutant ΔCgscd1 was obtained using homologous recombination. The ΔCgscd1 mutant showed no melanin accumulation on appressoria formation and vegetative hyphae. Furthermore, the virulence of ΔCgscd1 was significantly reduced in comparison with the wild-type (WT) strain. Further investigations showed that the growth rate as well as germination and appressorium formation of ΔCgscd1 displayed no difference compared to the wild-type and complemented transformant Cgscd1com strains. Furthermore, we found that the appressorial turgor pressure in the ΔCgscd1 mutant showed no difference compared to that in the WT and Cgscd1com strains in the incipient cytorrhysis experiment. However, fewer infectious hyphae of ΔCgscd1 were observed in the penetration experiments, suggesting that the penetration ability of nonpigmented appressoria was partially impaired. In conclusion, we identified the CgSCD1 gene, which is involved in melanin synthesis and pathogenicity, and found that the melanization defect did not affect appressorial turgor pressure in C. gloeosporioides.

https://www.mdpi.com/2076-0817/9/2/141/pdf

CgSCD1 Is Essential for Melanin Biosynthesis and Pathogenicity of Colletotrichum gloeosporioides.

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a 'brute-force' approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation 'naifu' invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora

Analysis of melanin biosynthesis in the plant pathogenic fungus Colletotrichum gloeosporioides.

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

https://hal.archives-ouvertes.fr/hal-03009610/document

Deciphering the Infectious Process of Colletotrichum lupini in Lupin through Transcriptomic and Proteomic Analysis

The ascomycete fungus Colletotrichum gloeosporioides infects a wide range of plant hosts and causes enormous economic losses in the world. The transcription factors (TFs) play an important role in development and pathogenicity of many organisms. In this study, we found that the C2H2 TF CgCrzA is localized in both cytoplasm and nucleus under standard condition, and it translocated from cytoplasm to nucleus in a calcineurin-dependent manner. Moreover, the ΔCgCrzA was hypersensitive to cell wall perturbing agents and showed severe cell wall integrity defects. Deletion of the CgCRZA inhibited the development of invasive structures and lost pathogenicity to plant hosts. Our results suggested that calcineurin-responsive TF CgCrzA was not only involved in regulating cell wall integrity, but also in morphogenesis and virulence in C. gloeosporioides.

Calcineurin-Responsive Transcription Factor CgCrzA Is Required for Cell Wall Integrity and Infection-Related Morphogenesis in Colletotrichum gloeosporioides.

In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite.IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea.

\n \n \n The gray mold fungus Botrytis cinerea (B. cinerea) infects a wide range of crops before and after harvest, causing huge losses worldwide. Inhibition mechanisms of B. cinerea in vitro and in plants by calcium propionate (CP), generally recognized as a safe substance, are described in this study.\n \n \n \n Wild-type and transgenic mutant strains of B. cinerea were used in the study to evaluate the effects of CP on fungal growth and development in vitro. Plant materials including tomato leaves and table grapes were tested for controlling efficiency of CP against gray mold deterioration in vivo.\n \n \n \n Mycelial growth of B. cinerea was inhibited by CP in a dose-dependent manner with occasional disruption of hyphal tips, causing cellular collapse and efflux of cell contents. Staining with fluorescein diacetate and propidium iodide indicated that CP decreased fungal cell viability. Inhibition efficiency of CP against B. cinerea was enhanced by reducing pH. In contrast, the vel1 mutant, which exhibited deficiency in acid production, was more resistant to CP, suggesting that inhibition of B. cinerea by CP is enhanced by the acidification ability of the fungus itself. Additionally, CP inhibited infection cushion development by germlings of B. cinerea. Infection assays with tomato leaves and table grapes showed that CP inhibited decay development in both host tissues. Moreover, application of CP on grapes 3 days prior to harvest could contribute to management of deterioration caused by spontaneous fungal diseases during storage.\n \n \n \n CP can suppress hyphal growth, inhibit infection cushion development, and reduce the virulence of B. cinerea. CP is thus promising for practical management of gray mold in fruit crops and merits further evaluation.\n

Inhibition of Botrytis cinerea and control of gray mold on table grapes by calcium propionate

N1-(2-chloro-4-pyridyl)-N3-phenylurea (CPPU), a synthetic cytokinin-active compound, is widely applied to induce parthenocarpic fruit set and enhance melon fruit enlargement (Cucumis melo L.). CPPU may also influence fruit quality; however, the mechanisms through which this occurs remain unknown. We investigated the differences in volatile emissions between parthenocarpic fruit set by CPPU (C) and seeded fruit set by artificial pollination (P). Gas chromatography–mass spectrometry (GC–MS) analysis revealed that six volatile organic compounds (VOCs) emitted by the P-group fruits were not detected in C-group fruits. The relative abundances of another 14 VOCs emitted by the CPPU-treated fruits were less than those in the P-group fruits. RNA sequencing analysis indicated that a total of 1027, 994, and 743 differentially expressed genes (DEGs) were detected in the C20 (treatment with 20 mg·L–1 CPPU) vs. P, P-C20 (pollination followed by 20 mg·L−1 CPPU treatment) vs. P, and P-C20 vs. C20 treatments, respectively. Compared with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, the DEGs related to fatty acid degradation and metabolism, which contribute to volatile production, were enriched. In particular, DEGs such as carotenoid cleavage dioxygenase (CCD)-, lipoxygenase (LOX)-, alcohol dehydrogenase (ADH)-, and alcohol acyltransferase (ATT)-related genes were closely related to the formation of volatiles. In summary, our study provides a metabolic and transcriptomic atlas, reveals the impact of CPPU on VOCs, and enhances our understanding of the mechanisms of CPPU that contribute towards generally reducing the quality of melon fruit.

Transcriptomic and Metabolomic Studies Reveal Mechanisms of Effects of CPPU-Mediated Fruit-Setting on Attenuating Volatile Attributes of Melon Fruit

Ethylene (ET) represents a signal that can be sensed by plant pathogenic fungi to accelerate their spore germination and subsequent infection. However, the molecular mechanisms of responses to ET in fungi remain largely unclear. In this study, Colletotrichum gloeosporioides was investigated via transcriptomic analysis to reveal the genes that account for the ET-regulated fungal development and virulence. The results showed that ET promoted genes encoding for fungal melanin biosynthesis enzymes, extracellular hydrolases, and appressorium-associated structure proteins at 4 h after treatment. When the germination lasted until 24 h, ET induced multiple appressoria from every single spore, but downregulated most of the genes. Loss of selected ET responsive genes encoding for scytalone dehydratase (CgSCD1) and cerato-platanin virulence protein (CgCP1) were unable to alter ET sensitivity of C. gloeosporioides in vitro but attenuated the influence of ET on pathogenicity. Knockout of the G-protein-coupled receptors CgGPCR3-1/2 and the MAPK signaling pathway components CgMK1 and CgSte11 resulted in reduced ET sensitivity. Taken together, this study in C. gloeosporioides reports that ET can cause transcription changes in a large set of genes, which are mainly responsible for appressorium development and virulence expression, and these processes are dependent on the GPCR and MAPK pathways.

/pdf/ethylene-promotes-expression-of-the-appressorium-and-25xhxq69.pdf

Ethylene Promotes Expression of the Appressorium- and Pathogenicity-Related Genes via GPCR- and MAPK-Dependent Manners in Colletotrichum gloeosporioides

Dandan Ren

Papers

CgSCD1 Is Essential for Melanin Biosynthesis and Pathogenicity of Colletotrichum gloeosporioides.

Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea.

Inhibition of Botrytis cinerea and control of gray mold on table grapes by calcium propionate

Transcriptomic and Metabolomic Studies Reveal Mechanisms of Effects of CPPU-Mediated Fruit-Setting on Attenuating Volatile Attributes of Melon Fruit

Ethylene Promotes Expression of the Appressorium- and Pathogenicity-Related Genes via GPCR- and MAPK-Dependent Manners in Colletotrichum gloeosporioides