The Role Of HSAF In The Interaction Between Lysobacter Enzymogenes And Neurospora Crassa

Lysobacter enzymogenes is a Gram-negative soil bacterium belonging to the genus Lysobacter and the Xanthomonadaceae family.As a new type of biocontrol bacteria,L.enzymogenes has great potential for application in the biological control of agricultural diseases and pharmaceutical treatments.Understanding the antifungal mechanism of L.enzymogenes is one of the prerequisites for its broad application.HSAF is one of the secondary metabolites of L.enzymogenes.Due to its antifungal effect,HSAF has received extensive attention.Though great efforts have been made to investigate the mode of action of HSAF,the specific antifungal mechanism of HSAF has been still unclear due to the lack of molecular,genetic and biochemical techniques developed for most studied fungi.Although a lot of research has been reported to study the mode of action of HSAF,the target of HSAF is still unknown and the cellular biological mechanism of HSAF has still not been fully elucidated.Neurospora crassa is the key organism in the history of genetics,biochemistry and molecular biology in the 20 th century.As a model organism,it is most commonly known for its role in proving the “one gene,one enzyme” hypothesis.Over the years,varieties of molecular,genetic and biochemical techniques and avant-garde tools developed for N.crassa,in particular,the availability of a full genome deletion strain set made N.crassa valuable as a classical genetic model to investigate a myriad of fundamental biological processes.In this study,we used the N.crassa as a model organism to study the antifungal mechanism of HSAF.We evaluated the antifungal effect of HSAF against N.crassa and performed RNA-Seq analysis to assess the genome-wide gene expression differences among different time points of the HSAF treatments.Based on transcriptome analysis,we consequently performed a systematic screening of 36 N.crassa deletion mutants to identify three genes with negative effects on HSAF resistance.We further used scanning electron microscopy(SEM)to observe the physical interaction between different L.enzymogenes OH11 and N.crassa mutants.In addition,we conducted WGCNA analysis to reveal that HSAF was able to trigger the autophagy pathway in N.crassa and further used confocal microscopy observation to verify this hypothesis.On the basis of our findings,we proposed a novel strategy for L.enzymogenes OH11 to infect its host prey.The specific results are as follows.Global transcriptional profiling combined with genetic and physiological analysis,spore germination,growth phenotype and differential gene expression analysis were performed.The experimental results showed that HSAF significantly inhibited the germination of N.crassa conidia,as well as the aerial hyphae growth in a dose-dependent manner,suggesting that N.crassa would be a good model to study the antifungal mechanism of HSAF.RNA-seq analysis showed that HSAF treatment led to increased expression of about 36 cell wall-related genes including seven chitin synthase genes and thicker cell walls.Screening of N.crassa gene deletion mutants combined with scanning electron microscopic observation revealed that three fungal cell wall integrity-related genes played an important role in the interaction between N.crassa and L.enzymogenes.These reports suggested that HSAF treatment could promote an antifungal resistance response in fungal cells.In addition,Weighted Gene Co-Expression Network Analysis(WGCNA),accompanied by confocal microscopy observation revealed that HSAF could trigger autophagy-mediated degradation and eventually result in cell death in N.crassa.The findings of this work provided new insights into the interactions between the predatory Lysobacter and its fungal prey.In conclusion,our results suggested that HSAF might interact with the fungal cell wall to assist L.enzymogenes OH11 in entering the fungal cells,thereby releasing more HSAF to cause autophagy-mediated degradation and eventually lead to fungal cell death.The findings of this study provided new perceptions about how L.enzymogenes interacted with its fungal prey and ultimately kill it.