Regulation Mechanisms Of Ethylene And Light In Growth, Development, And Virulence Of Botrytis Cinerea

Botrytis cinerea is one of the most important necrotrophic plant pathogens, jeopardizing a wide range of host crops, especially fruits and vegetables which provide human beings with essential nutrients. However, the supply of fruits and vegetables usually suffers from huge losses worldwide, mainly due to the destructive postharvest pathogen attack. The knowledge focused on the effect of environmental factors on the growth, development and, pathogenicity of fungal pathogens would most probably contribute to the development of strategies to control postharvest diseases.Ethylene （ET） is an important plant hormone capable of influencing maturity, softening, and commercial quality of various postharvest crops. However, ET sensing and production in pathogenic fungi are less recognized, and both the role and origin of the disease ET in pathosystems are obscure. Light is another important environment factor which has considerable regulatory effects over fungal species, however the significance of light in postharvest circumstances is undervalued. This work used B. cinerea as the object to study the regulation mechanisms of ethylene and light signal in growth, development, and virulence of the fungus. Based on this study, some potential strategies aiming to control postharvest diseases have been proposed. The summary of this study is listed below:1. To clarify whether fungal pathogen contributes to ET production in plant pathosystems, the difference of ET production between plant and B. cinerea was taken into account:ET producing rate in B. cinerea is significantly enhanced by light, while ET production in fruits is not. In this part of study, the ET production rate of the fruit-B. cinerea pathosystems in light was found to be almost twice amount of that in dark, indicating this increased ET production should most probably originate from the ET produced by B. cinerea, which was thus defined as Pathogen’s ET in the pathosystem. This procedure to detect Pathogen’s ET seems to be practical enough to be developed into a marker to timely diagnose fungal diseases in postharvest fruits and vegetables.2. Further study concentrated on the effect of exogenous ET on B. cinerea, and found that ET could enhance the haphal differentiation and appressorium formation, as well as induce the expression of polygalacturonase genes, indicating that the growth, development and virulence of B. cinerea could be affected by ET.3. Since previous work found that B. cinerea produced ET in vitro using methionine （Met） as precursor, and ET could also affect its growth and expression of pathogenicity factors, the RNA-seq was subsequently employed to compare the transcriptome of B. cinerea as influenced with addition of either Met or ET. The genes correlated with ET biosynthesis and sensing in B. cinerea were systemically digged out and analyzed. These information established foundation for further understanding the mechanisms of ET production and sensing in this fungus.4. Among the differentially expressed genes obtained from transcriptome analysis, one Met-upregulated gene coding for aspartate transaminase （beast, BC1G₀1276） was used for further investigation. Its transcription was highly induced during infection procedure. The knock-out mutant strain created by homologous recombination showed slightly reduced growth rate without any significant alteration in colony morphology. However, the mutant strain achieved stronger ET production rate than wild type strain, and unexpectedly, the mutant strain could even produce ET on the minimum medium without the supplement of Met. Furthermore, the mutant strain lost secretion ability of reactive oxygen species （ROS） and pathogenicity on integrated host tissues. These results indicate that beast is a newly recognized virulence factor of B. cinerea, but the pathogenicity is probably independent of the extent of its ET-producing ability.5. Since light can not only enhance ET production in B. cinerea, but also directly affected growth, development, and pathogenicity of this pathogen, the effect of light on B. cinerea was subsequently investigated. Preliminary screening showed that different light signal had various regulative effects on B. cinenea strains, with two phenomena being most conserved:green light could retard hyphal growth rate, and blue light could inhibit sporulation in B. cinerea. In addition, the UV resistance of B. cinerea was dependent on exposure to light, however, the knock-out mutant of the homologue of White collar 1 in B. cinerea, Δbcwc1, was hypersensitivity to UV irrespective of light or dark treatment. This pattern was confirmed in Fusarium oxysporum and Alternaria alternate as well, which then arose to optimize the UV treatment to control postharvest diseases:UV application during night period would acquire much better fungicidal efficacy than application during day period. These findings provided solid morphological patterns for light sensing study in B. cinerea, as well as potential improvement for technical control strategies against this pathogen.6. The mutant strain Δbcwcl also showed increased ET production rate, and blue light was found to better enhance ET production rate in B. cinerea, both indicating that the blue light signaling and ET production pathways were correlated in this fungal pathogen. These results lead to a novel breakthrough point for further clarifying the regulative mechanisms of fungal ET biosynthesis.7. The human fungal pathogen Cryptococcus neoformans was investigated as a reference object to understand the regulation mechanisms of light signal in pathogenic fungi. Homolog of the conserved fungal blue light photo receptor gene wc-1 in C neoformans, the BWC1, is involved in UV sensitivity, repression of mating in response to light, and required for virulence of this pathogen. Replacing the conserved chromophore binding site in BWC1 by point mutation resulted in deprivation of all light sensing responses of C. neoformans, but didn’t affect the virulence, indicating that light sensing is dispensable during pathogenesis of this fungus. Further transcriptome analysis comparing among the wild type, knock-out mutant, and chromorphore-binding site mutant strains ruled out the differentially expressed gene affected by light, and obtained those potential virulence genes regulated by Bwcl which need to be further clarified.