| The ascomycete Botrytis cinerea is a plant pathogenic fungus with a necrotrophic life style which causes gray mold on more than 200 plant species,many of which are economically important crops including vegetables,ornamentals,bulbs,and fruits.It is worthwhile to study about this fungus due to its broad host range and huge economic loss.B.cinerea kills the host cells through production of toxins and reactive oxygen species as well as inducing oxidative burst in plant.It has been suggested that this fungus causes brutal damage of host tissue through massive secretion of toxic substances.Two groups of nonspecific toxins have been identified and in this fungus;the botryanes and the botcinins.Our laboratory has previously obtained knock-out mutants of melanin biosynthesis related enzymes,including ?bcpks13(polyketide synthase coding gene),?bcbrn1(tetrahydroxynaphthalene(T4HNR)coding gene),and ?bcscd1(scytalone dehydratase coding gene).Among these mutants,?bcbrn1 and ?bcpks13 showed high virulence phenotype,whilst ?bcscd1 showed a significant decrease in virulence.The results suggested that melanin biosynthesis related enzymes and intermediates play a special role in virulence of B.cinerea.According to previous data and transcriptome analysis,the expression of all the genes related to toxin production significantly decreased in ?bcscd1 compared to wild type.This study is focusing on ?bcscd1 and its toxin production because of the considerable reduction of virulence factors in this mutant.Any findings that can help to reduce the incidence of gray mold diseases are beneficial for the control of this aggressive fungus.Therefore,this study analyzed in vitro production of toxins in this ?bcscd1 mutant as well as ?bcpks13 and WT.The main results are listed below:1.To analyze the disease phenotype of three melanin biosynthesis-deficient mutants,we performed a virulence assay using Arabidopsis thaliana and tomato leaves which were inoculated with the mycelium discs of WT and ?bcpks13,?bcbrn1,and ?bcscd1.As expected,leaves inoculated with ?bcpks13 and ?bcbrn1 showed more expanded lesion area in comparison to leaves inoculated with wild type(WT).In addition,disease symptoms appeared sooner in these two mutants.On the other hand,leaves inoculated with ?bcscd1 displayed limited lesion area compared to that of wild type and moreover,disease symptoms appeared later in this mutant.This assay aimed to perform a comparison between the disease symptoms resulting from inoculation of mycelium and the disease symptoms resulting from application of toxin.2.To analyze toxin production in WT,?bcscd1,and ?bcpks13,we performed a liquid-liquid extraction(LLE)from the culture of these mutants and WT to obtain crude toxins.Application of toxin on tomato leaves demonstrated no direct relationship between toxin production and reduced virulence of ?bcscd1 or increased virulence of ?bcpks13.Interestingly,the results showed that toxicity of crudely extracted toxin from ?bcpks13 is less than toxicity of ?bcscd1 which is in contrary to the result from virulence assay.3.To identify changes in the transcript of WRKY33 during application of toxins,the expression profile of WRKY33 was investigated.WRKY33 is a key transcription factor in pathogen-induced defense signaling and that is essential for defense toward Botrytis cinerea.The results were in accordance to the observed phenotype.The expression of WRKY33 decreased upon application of toxins from ?bcpks13 and increased upon application of toxins from both WT and ?bcscd1.Taken together,the relationship between toxin production and decreased or increased virulence in melanin biosynthesis-deficient mutants was analyzed.The results suggested that toxicity of extracted toxin has no relationship between induction of virulence in ?bcpks13 or reduced virulence in ?bcscd1. |
