The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of ΔCfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development (CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation (CfCBP1 and CfCHS7), pectin degradation (CfPelA and CfPelB), sexual development (CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis (CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

1,8-Dihydroxynaphthalene Melanin Biosynthesis in Colletotrichum fructicola Is Developmentally Regulated and Requires the Cooperative Function of Two Putative Zinc Finger Transcription Factors.

In ascomycetes, 1,8-dihydroxynaphthalene (DHN) melanin plays important protective functions and its production is usually coupled with development and environmental stress responses. The regulation of melanin biosynthesis, however, remains obscure. Colletotrichum fructicola is a phytopathogen with a broad host range that produces melanized appressoria and perithecia. In this study, we annotated melanin genes in a high-quality C. fructicola genome and characterized two zinc finger transcription factors (TFs) (cmr1 and cmr2) that form a loosely organized gene cluster with several melanin biosynthesis genes. Deleting either TF abolished melanization in both mycelia and perithecia but did not affect appressoria. The deletion mutants also showed perithecial development defects. Overexpressing cmr1 in Δcmr2 strongly activated the expression of melanin biosynthesis genes including pks1, scd1, t4hr1, and thr1 and caused hyper-accumulation of charcoal to black pigment(s). On the other hand, overexpressing cmr2 in Δcmr1 activated pks1, t4hr1, and thr1, but not scd1. We conclude that proper DHN melanin accumulation in C. fructicola requires the cooperative function of two in-cluster TFs that also regulate perithecial development. The study clarifies DHN melanin regulations in C. fructicola and expands the function of melanin in-cluster TFs to sex regulation.

Transcription Factor CfSte12 of Colletotrichum fructicola Is a Key Regulator of Early Apple Glomerella Leaf Spot Pathogenesis.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a rapidly emerging disease leading to defoliation, fruit spot, and storage fruit rot on apple in China. Little is known about the mechanisms of GLS pathogenesis. Early transcriptome analysis revealed that expression of the zinc finger transcription factor Ste12 gene in C. fructicola (CfSte12) was upregulated in appressoria and leaf infection. To investigate functions of CfSte12 during pathogenesis, we constructed gene deletion mutants (ΔCfSte12) by homologous recombination. Phenotypic analysis revealed that CfSte12 was involved in pathogenesis of nonwounded apple fruit and leaf, as well as wounded apple fruit. Subsequent histological studies revealed that loss of pathogenicity by ΔCfSte12 on apple leaf was expressed as defects of conidium germination, appressorium development, and appressorium-mediated penetration. Further RNA sequencing-based transcriptome comparison revealed that CfSte12 modulates the expression of genes related to appressorium function (e.g., genes for the tetraspanin PLS1, Gas1-like proteins, cutinases, and melanin biosynthesis) and candidate effectors likely involved in plant interaction. In sum, our results demonstrated that CfSte12 is a key regulator of early apple GLS pathogenesis in C. fructicola In addition, CfSte12 is also needed for sexual development of perithecia and ascospores.IMPORTANCE Glomerella leaf spot (GLS) is an emerging fungal disease of apple that causes huge economic losses in Asia, North America, and South America. The damage inflicted by GLS manifests in rapid necrosis of leaves, severe defoliation, and necrotic spot on the fruit surface. However, few studies have addressed mechanisms of GLS pathogenesis. In this study, we identified and characterized a key pathogenicity-related transcription factor, CfSte12, of Colletotrichum fructicola that contributes to GLS pathogenesis. We provide evidence that the CfSte12 protein regulates many important pathogenic processes of GLS, including conidium germination, appressorium formation, appressorium-mediated penetration, and colonization. CfSte12 also impacts development of structures needed for sexual reproduction which are vital for the GLS disease cycle. These results reveal a key pathogenicity-related transcription factor, CfSte12, in C. fructicola that causes GLS.

Comparative analysis of the mitochondrial genomes of Colletotrichum gloeosporioides sensu lato: insights into the evolution of a fungal species complex interacting with diverse plants.

BACKGROUND: The fungal species complex Colletotrichum gloeosporioides sensu lato contains over 20 plant-interacting species. These species exhibit different life styles (e.g., endophytes, foliar and fruit pathogens) and show considerable variation in host and tissue adaptation strategies. Accurate species delimitation in C. gloeosporioides s.l. is very challenging due to nascent lineage boundaries and phenotypic plasticity, which strongly impedes studies of the complex's host-interaction biology. In this study, we first sequenced and compared nine mitogenomes belonging to four C. gloeosporioides s.l. species lineages (C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense s.l.), and evaluated the usefulness of mitogenome sequence in complementing prevailing nuclear markers for species delimitation. RESULTS: The C. gloeosporioides s.l. mitogenomes ranged between 52,671 and 58,666 bp in size, and each contained an identical set of genes transcribed in the same direction. Compared with previously reported Colletotrichum mitogenomes, these mitogenomes were uniquely featured by: (1) significantly larger genome size due to richer intron content and longer intergenic region; (2) striking GC content elevation at the intergenic region; and (3) considerable intron content variation among different species lineages. Compared with nuclear DNA markers commonly used in phylogeny, the mitogenome nucleotide diversity was extremely low, yet the mitogenome alignment contained the highest number of parsimony informative sites, which allowed the generation of a high-resolution phylogeny recognizing all taxonomic lineages, including ones belonging to the very nascent C. siamense s.l. complex. The tree topology was highly congruent with the phylogeny based on nuclear marker concatenation except for lineages within C. siamense s.l. Further comparative phylogenetic analysis indicated that lineage-specific rapid divergence of GS and SOD2 markers confounded concatenation-based species relationship inference. CONCLUSIONS: This study sheds light on the evolution of C. gloeosporioides s.l. mitogenomes and demonstrates that mitogenome sequence can complement prevailing nuclear markers in improving species delimitation accuracy. The mitogenome sequences reported will be valuable resources for further genetic studies with C. gloeosporioides s.l. and other Colletotrichum species.

CfHMG Differentially Regulates the Sexual Development and Pathogenicity of Colletotrichum fructicola Plus and Minus Strains.

Colletotrichum fructicola shows morphological and genetic differences in plus and minus strains. However, the mechanism of the differentiation between two types of strains is still largely unclear. Our early transcriptome analysis revealed that CfHMG expression differed in plus and minus strains. To define the functions of the CfHMG gene, we constructed gene deletion mutants by homologous recombination. We found that a CfHMG deletion mutant of the minus strain, CfHMG-M, could lead to a reduction in perithecium sizes and densities on media and sterile perithecium formation compared with the minus wild type (WT), whereas there was no effect for the plus mutant CfHMG-P. In co-cultures between CfHMG-P and minus WT, CfHMG-M and plus WT, or CfHMG-P and CfHMG-M, the quantities of perithecia were all reduced significantly. When conidial suspensions were inoculated on non-wounded apple fruit, it was found that the virulence of the minus mutant decreased significantly but not for the plus one. Further, we found that the virulence decrease in minus mutants was caused by a decrease in the conidium germination rate. Our results indicate that CfHMG of C. fructicola plays an important role in the mating line formation between the plus and minus strain for both strains and differentially regulates the perithecium size, density, fertilization, and virulence of the minus strain. The results are significant for further detecting the differentiated mechanisms between the plus and minus strains in Colletotrichum fungi.

The MAP Kinase CfPMK1 Is a Key Regulator of Pathogenesis, Development, and Stress Tolerance of Colletotrichum fructicola.

The Ascomycetes fungus Colletotrichum fructicola causes severe diseases on a wide range of crops, fruits, and vegetables. Its pathogenic mechanisms, however, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are conserved regulators of fungal development and pathogenesis. In this study, a Fus3/Kss1-related MAPK from C. fructicola was functionally characterized via gene deletion. On potato dextrose agar (PDA) and oatmeal agar media, the CfPMK1 gene deletion mutants (ΔCfPMK1) were slightly reduced in radial growth rate, severely limited in aerial hyphal differentiation and hyphal melanization, and formed deformed perithecia that were smaller in size and more compactly organized relative to wild type. When artificially inoculated on plants, conidia of these mutants failed to differentiate appressoria or penetrate cuticle, and their pathogenicity defect could not be rescued by wounding plant tissue prior to inoculation. On PDA, ΔCfPMK1 mutants were hypersensitive to osmotic stresses, but were more tolerant to membrane and cell wall stresses. Genetic complementation rescued all phenotypic changes associated with CfPMK1 gene deletion. Based on GFP fusion expression, CfPMK1 protein accumulation was detected at all life stages, and the accumulation level was higher in nascent appressoria relative to conidia. Overall, this study identified CfPMK1 as a key regulator of appressorium and sexual development, pathogenesis, and stress tolerance in C. fructicola.