A Single-Nucleotide Deletion in the Transcription Factor Gene bcsmr1 Causes Sclerotial-Melanogenesis Deficiency in Botrytis cinerea

Botrytis cinerea is an important plant pathogenic fungus with a wide range of host. It usually produces black-colored sclerotia (BS) due to deposition of 1,8-dihydroxynaphthalene melanin in sclerotial melanogenesis. Our previous study (Zhou et al., 2018) reported six B. cinerea isolates producing orange-colored sclerotia (OS) with deficiency in sclerotial melanogenesis. Comparison of ecological fitness (conidia, mycelia, sclerotia), natural distribution, and melanogenesis of selected BS and OS isolates suggests that sclerotia play an important role in the disease cycle caused by B. cinerea. However, the molecular mechanism for formation of the OS B. cinerea remains unknown. This study was done to unravel the molecular mechanism for the sclerotial melanogenesis deficiency in the OS isolates. We found that all the five sclerotial melanogenesis genes (bcpks12, bcygh1, bcbrn1/2, bcscd1) were down-regulated in OS isolates, compared to the genes in the BS isolates. However, the sclerotial melanogenesis-regulatory gene bcsmr1 had similar expression in both types of sclerotia, suggesting the sclerotial melanogenesis deficiency is due to loss-of-function of bcsmr1, rather than lack of expression of bcsmr1. Therefore, we cloned bcsmr1 from OS (bcsmr1OS) and BS (bcsmr1BS) isolates, and found a single-nucleotide deletion in bcsmr1OS. The single-nucleotide deletion caused formation of a premature stop codon in the open reading frame of bcsmr1OS, resulting in production of a 465-aa truncated protein. The transcription activity of the truncated protein was greatly reduced, compared to that of the 935-aa full-length protein encoded by bcsmr1BS in the BS isolates. The function of bcsmr1OS was partially complemented by bcsmr1BS. This study not only elucidated the molecular mechanism for formation of orange-colored sclerotia by the spontaneous mutant XN-1 of B. cinerea, but also confirmed the regulatory function of bcsmr1 in sclerotial melanogenesis of B. cinerea.

[1]  Weidong Chen,et al.  Contrast Between Orange- and Black-Colored Sclerotial Isolates of Botrytis cinerea: Melanogenesis and Ecological Fitness. , 2017, Plant disease.

[2]  A. Casadevall,et al.  Functions of fungal melanin beyond virulence. , 2017, Fungal biology reviews.

[3]  Xiang-ming Xu,et al.  Vayg1 is required for microsclerotium formation and melanin production in Verticillium dahliae. , 2017, Fungal genetics and biology : FG & B.

[4]  M. Ambrico.  SPECIAL ISSUE: Melanin, a long lasting history bridging natural pigments and organic bioelectronics , 2016 .

[5]  J. Schumacher,et al.  Light governs asexual differentiation in the grey mould fungus Botrytis cinerea via the putative transcription factor BcLTF2. , 2016, Environmental microbiology.

[6]  G. S. de Hoog,et al.  The role of melanin pathways in extremotolerance and virulence of Fonsecaea revealed by de novo assembly transcriptomics using illumina paired-end sequencing , 2016, Studies in mycology.

[7]  J. Schumacher.  DHN melanin biosynthesis in the plant pathogenic fungus Botrytis cinerea is based on two developmentally regulated key enzyme (PKS)‐encoding genes , 2016, Molecular microbiology.

[8]  Y. Elad,et al.  Plant Hosts of Botrytis spp. , 2016 .

[9]  D. Jiāng,et al.  CmpacC regulates mycoparasitism, oxalate degradation and antifungal activity in the mycoparasitic fungus Coniothyrium minitans. , 2015, Environmental microbiology.

[10]  Pinkuan Zhu,et al.  Loss of bcbrn1 and bcpks13 in Botrytis cinerea Not Only Blocks Melanization But Also Increases Vegetative Growth and Virulence. , 2015, Molecular plant-microbe interactions : MPMI.

[11]  Kylie J. Boyce,et al.  The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei , 2015, PloS one.

[12]  Maximilian Wenderoth,et al.  Alternaria alternata transcription factor CmrA controls melanization and spore development. , 2014, Microbiology.

[13]  D. Jiāng,et al.  Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum. , 2014, Environmental microbiology.

[14]  Noriko Kinoshita,et al.  Genome-wide single nucleotide polymorphisms and insertion–deletions of Oryza sativa L. subsp. japonica cultivars grown near the northern limit of rice cultivation , 2014, Molecular Breeding.

[15]  J. Schumacher,et al.  The Transcription Factor BcLTF1 Regulates Virulence and Light Responses in the Necrotrophic Plant Pathogen Botrytis cinerea , 2014, PLoS genetics.

[16]  M. Hahn,et al.  Involvement of two type 2C protein phosphatases BcPtc1 and BcPtc3 in the regulation of multiple stress tolerance and virulence of Botrytis cinerea. , 2013, Environmental microbiology.

[17]  Qianqian Yang,et al.  Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development, Virulence and Multi-Stress Tolerance in Botrytis cinerea , 2013, PloS one.

[18]  S. Fillinger,et al.  A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET , 2013, PloS one.

[19]  Qianqian Yang,et al.  Involvement of BcVeA and BcVelB in regulating conidiation, pigmentation and virulence in Botrytis cinerea. , 2013, Fungal genetics and biology : FG & B.

[20]  J. Schumacher,et al.  Natural Variation in the VELVET Gene bcvel1 Affects Virulence and Light-Dependent Differentiation in Botrytis cinerea , 2012, PloS one.

[21]  Yangrae Cho,et al.  Transcription Factor Amr1 Induces Melanin Biosynthesis and Suppresses Virulence in Alternaria brassicicola , 2012, PLoS pathogens.

[22]  S. Fillinger,et al.  The osmosensing signal transduction pathway from Botrytis cinerea regulates cell wall integrity and MAP kinase pathways control melanin biosynthesis with influence of light. , 2011, Fungal genetics and biology : FG & B.

[23]  C. Tanaka,et al.  A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea , 2010, Molecular Genetics and Genomics.

[24]  S. Fillinger,et al.  Fungicide-Driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis cinerea , 2009, PLoS pathogens.

[25]  S. Georgeault,et al.  Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia , 2009, BMC Microbiology.

[26]  C. Tanaka,et al.  Characterization of the BMR1 gene encoding a transcription factor for melanin biosynthesis genes in the phytopathogenic fungus Bipolaris oryzae. , 2008, FEMS microbiology letters.

[27]  Brian Williamson,et al.  Botrytis cinerea: the cause of grey mould disease. , 2007, Molecular plant pathology.

[28]  B. Horwitz,et al.  Melanin Biosynthesis in the Maize Pathogen Cochliobolus heterostrophus Depends on Two Mitogen-Activated Protein Kinases, Chk1 and Mps1, and the Transcription Factor Cmr1 , 2007, Eukaryotic Cell.

[29]  C. J. Rodrigues,et al.  Appressorium turgor pressure of Colletotrichum kahawae might have a role in coffee cuticle penetration , 2004, Mycologia.

[30]  R. P. Doss,et al.  Melanin in the extracellular matrix of germlings of Botrytis cinerea. , 2003, Phytochemistry.

[31]  S. Ferrone,et al.  A Single-nucleotide Deletion Leads to Rapid Degradation ofTAP-1 mRNA in a Melanoma Cell Line* , 2003, The Journal of Biological Chemistry.

[32]  D. Debieu,et al.  Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. , 2002, Pest management science.

[33]  B. Gómez,et al.  Melanins in fungal pathogens. , 2002, Journal of medical microbiology.

[34]  I. Furusawa,et al.  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 , 2000, Molecular microbiology.

[35]  E. Jacobson.  Pathogenic roles for fungal melanins. , 2000, Clinical microbiology reviews.

[36]  M. Persuy,et al.  A single nucleotide deletion resulting in a premature stop codon is associated with marked reduction of transcripts from a goat beta-casein null allele. , 1999, Animal genetics.

[37]  M. Butler,et al.  Fungal melanins : a review , 1998 .

[38]  F. Chumley,et al.  Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis. , 1998, Molecular plant-microbe interactions : MPMI.

[39]  R. Howard,et al.  Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. , 1996, Annual review of microbiology.

[40]  M. Wheeler,et al.  Biosynthesis and Functions of Fungal Melanins , 1986 .

[41]  H. Buchenauer,et al.  Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea , 1985 .

[42]  Y. Kubo.  Effect of Tricyclazole on Appressorial Pigmentation and Penetration from Appressoria ofColletotrichum lagenarium , 1982 .