RESUMEN
The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.
Asunto(s)
Botrytis/genética , Botrytis/patogenicidad , Ceruloplasmina/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Hierro/metabolismo , Botrytis/enzimología , Ceruloplasmina/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Hojas de la Planta/microbiología , Esporas Fúngicas , Virulencia/genéticaRESUMEN
A halotolerant endoglucanase with a molecular mass of 39â¯kDa was obtained from the solid fermentation of sugarcane bagasse by the fungus Botrytis ricini URM 5627 and isolated using only two purification processes: fractionation with ammonium sulphate and size-exclusion chromatography resulting in an activity of 1289.83â¯U/mL. After the isolation, biochemical characterizations were performed, giving a temperature of 50⯰C and optimum pH of 5. The enzyme was stable at 39-60⯰C for 60â¯min and at a pH of 4-6. The enzymatic activity increased in the presence of Na+, Mn2+, Mg2+ and Zn2+ and decreased in the presence of Ca2+, Cu2+, and Fe2+. The endoglucanase revealed a halotolerant profile since its activity increased proportionally to an increase in NaCl concentration. The maximum activity was reached at 2â¯M NaCl with a 75% increase in activity. The enzyme had a Km of 0.1299⯱â¯0.0096â¯mg/mL and a Vmax of 0.097⯱â¯0.00121â¯mol/min/mL. During application in saccharification tests, the enzyme was able to hydrolyse sugarcane bagasse, rice husk, and wheat bran, with the highest production of reducers/fermentable sugars within 24â¯h of saccharification for wheat bran (137.21â¯mg/g). Therefore, these properties combined make this isolated enzyme a potential candidate for biotechnological and industrial applications.
Asunto(s)
Botrytis/enzimología , Celulasa/metabolismo , Fibras de la Dieta , Estabilidad de Enzimas , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Saccharum/metabolismo , TemperaturaRESUMEN
The ascomycete plant pathogen Botrytis cinerea secretes aspartic proteinase (AP) activity. Functional analysis was carried out on five aspartic proteinase genes (Bcap1-5) reported previously. Single and double mutants lacking these five genes showed neither a reduced secreted proteolytic activity, nor a reduction in virulence and they showed no alteration in sensitivity to antifungal proteins purified from grape juice. Scrutiny of the B. cinerea genome revealed the presence of nine additional Bcap genes, denoted Bcap6-14. The product of the Bcap8 gene was found to constitute up to 23% of the total protein secreted by B. cinerea. Bcap8-deficient mutants secreted approximately 70% less AP activity but were just as virulent as the wild-type strain. Phylogenetic analysis showed that Bcap8 has orthologs in many basidiomycetes but only few ascomycetes including the biocontrol fungus Trichoderma harzanium. Potential functions of the 14 APs in B. cinerea are discussed based on their sequence characteristics, phylogeny and predicted localization.
Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Botrytis/enzimología , Proteínas Fúngicas/metabolismo , Secuencia de Aminoácidos , Antifúngicos/farmacología , Proteasas de Ácido Aspártico/clasificación , Proteasas de Ácido Aspártico/genética , Botrytis/efectos de los fármacos , Botrytis/genética , Botrytis/patogenicidad , Clonación Molecular , Citosol/enzimología , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/farmacología , Alineación de SecuenciaRESUMEN
The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.
Asunto(s)
Ácido Abscísico/genética , Ácido Abscísico , Ácido Abscísico/uso terapéutico , Botrytis/enzimología , Botrytis/metabolismo , Ascomicetos/enzimología , Petunia/genética , Retroelementos/genética , Secuencias Repetidas TerminalesRESUMEN
Four phytopathogenic fungi were cultivated up to six days in media containing chitooligosaccharide mixtures differing in average DP and FA. The three different mixtures were named Q3 (which contained oligosaccharides of DP2-DP10, with DP2-DP7 as main components), Q2 (which contained oligosaccharides of DP2-DP12, with DP2-DP10 as main components) and Q1 (which derived from Q2 and contained oligomers of DP5-DP8 with hexamer and a heptamer as the main components). The novel aspect of this work is the description of the effect of mixtures of oligosaccharides with different and known composition on fungal growth rates. The growth rate of Alternaria alternata and Rhizopus stolonifer was initially inhibited by Q3 and Q2 at higher concentrations. Q1 had a growth stimulating effect on these two fungi. Growth of Botrytis cinerea was inhibited by Q3 and Q2, while Q1 had no effect on the growth of this fungus. Growth of Penicillium expansum was only slightly inhibited by higher concentrations of sample Q3, while Q2 and Q1 had no effect. The inhibition of growth rates or their resistance toward chitooligosaccharides correlated with the absence or presence of chitinolytic enzymes in the culture media, respectively.
Asunto(s)
Alternaria/crecimiento & desarrollo , Botrytis/crecimiento & desarrollo , Quitina/metabolismo , Oligosacáridos/metabolismo , Penicillium/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Rhizopus/crecimiento & desarrollo , Acetilación , Alternaria/enzimología , Botrytis/enzimología , Quitina/análogos & derivados , Medios de Cultivo , Frutas/microbiología , Cinética , Espectrometría de Masas , Oligosacáridos/química , Penicillium/enzimología , Rhizopus/enzimologíaRESUMEN
The antifungal activity on Botrytis cinerea of the diterpenoids 3beta-hydroxy-kaurenoic acid and kaurenoic acid, obtained from the resinous exudates of Pseudognaphalium vira vira, was determined. 3beta-Hydroxy-kaurenoic acid reduced the mycelial growth of B. cinerea in solid and liquid media. Additionally, the damage produced by the fungus on the surface of tomato leaves in the presence of the diterpenoids was evaluated. A higher protective effect was observed in the presence of the hydroxylated diterpene. On the other hand, the effect of the diterpenoids on the production of enzymes that participate in the plant infection by B. cinerea was analyzed. p-Nitrophenylbutyrate esterase production was induced by both diterpenoids, whereas laccase production was only induced by the hydroxylated diterpene. In the study of the mechanism of action of these compounds, it was determined that 3beta-hydroxy-kaurenoic acid would produce permeabilization of the cell membrane of B. cinerea.