Study On The Interspecific Laccase Induction Between Different White Rot Fungi Based On Transcriptional,Protein And Metabolic Level

Interspecific interactions between white rot fungi are always accompanied by competition for territory and resources to maintain their own growth.When the niche of different fungal species overlaps,fungal mycelium contacts with each other and trigger a series of antagonistic responses in the interaction zone,including changes in mycelial morphology,production of extracellular enzymes and secretion of secondary metabolites.These responses can play an important role in enhancing attack or defense system and ability of nutrient absorption of fungi to gain advantage in the competition.Previous studies have shown that the laccase activity in the mycelial interaction zone was increased when different white rot fungi co-cultured on the solid medium.In order to investigate the interaction mechanism,especially the mechanism of laccase induction between different white rot fungi species,three white rot fungi Trametes versicolor(Tv),Pleurotus ostreatus(Po)and Dichomitus squalens(Ds)were selected for this study,because their laccase activity can be efficiently induced during co-culture with other white rot fungi.In addition,transcriptomics,metabolomics and proteomics techniques and other related verification experiments were used to analyze the antagonistic mechanism among different white rot fungi species and explore the metabolic and regulated network of interspecific laccase induction based on transcriptional,protein and metabolic level.In this paper,NBT staining method was used to detect the accumulation of reactive oxygen species in the interaction zones and edge zones in the co-cutures among three white rot fungi species.The results showed that more reactive oxygen species(ROS)accumulated in the mycelial interaction zone in the three fungal co-cultures(Tv Po,Po Ds,and Tv Ds)compared to edge of single fungi hyphae.In addition,scanning electron microscopy observation revealed that compared to the regular and smooth tubular structure of hypha in mono-cultures,hyphal morphology in the interaction zone of co-cultures was damaged to different extent,and the hyphae were tightly intertwined,uneven,clumpy and disaggregated.Notably,spores production can be clearly observed in the hyphae of Ds mono-culture,while spore-free hyphae in the interaction zones of Tv Ds and Po Ds co-cultures,which showed that a large amount of ROS is likely to be generated during the interspecific interaction,thereby changing the shape of the hyphae and inhibiting the production of fungal spores by destroying fungal cell membrane and demaging DNA and protein in the cell.In order to further confirm that interspecific laccase-inducing phenomenon of white rot fungi is related to oxidative stress,RNA-seq sequencing was used to obtain the gene expression profile of three co-cultures(Tv Po,Po Ds,and Tv Ds)and three mono-cultures(Tv,Po and Ds).The result showed that a large number of genes resistant to oxidative stress were up-regulated in the three co-cultures.These genes mainly encode aldo/keto reductase,glutathione Stransferase,laccase,manganese peroxidase,multi-drug resistant ABC transporter,alcohol dehydrogenase and aldehyde dehydrogenase,etc.These proteins play a similar function to remove ROS and detoxify xenobiotics.In addition,there are also some up-regulated genes encoding proteins which are related to generation of oxidative stress,such as alcohol oxidase,NAD(P)-binding protein and cytochrome P450.Up-regulation of these gene expressions will disrupt the dynamic balance of ROS and increase the accumulation of ROS in fungal cells..This paper focuses on the identification of laccase genes that were significantly up-regulated in co-cultures.The results showed that the laccase genes lcc5 and lcc11 in Ds,the laccase genes lacc2 and lacc10 in Po,and the lacase genes lac3 and lac1 in Tv were significantly up-regulated during the interaction with the other two fungi,and q RT-PCR also verified the expression changes of these laccase genes and other genes responding to oxidative stress,which confirmed the reliability of the RNA-seq data.Analysis of functions annotated by these highly expressed laccase genes and other significantly up-regulated genes revealed that laccase induction is likely to be the result of defense response to oxidative stress and competition for nutrients and territory with competitors.This result is also consistent with our previous biological data of laccase induction.In addition,this is first paper trying to fing the key proteins showing common or specific responses under the interactions among three white rot fungi Tv,Po and Ds by using proteomics technology and also to reveal the laccase induction and antagonistic mechanism of white rot fungi under co-culture condition.When comparing the proteomic data of white rot fungi under co-culture and mon-culture conditions,we found that a large number of proteins were upregulated in the three co-cultures(Tv Po,Po Ds,and Tv Ds)and involved in the antioxidant response system and antifungal antagonistic mechanism of nutrition competition.Among these up-regulated differentially expressed proteins,the largest group was antioxidant-related proteins,followed by carbohydrate metabolism-related proteins and energy production-related proteins.Although there were some differences in the antioxidant response system in this three co-cultures,the protein expression of laccase,catalase,and peroxidase was significantly upregulated in all co-cultures compared to the mono-cultures.In addition,three co-cultures also showd common enhance antioxidant defense response systems including aldo/keto reductase,superoxide dismutase,thioredoxin,thioredoxin reductase,and glutathione thiotransferase.These up-regulated proteins also provide important clues for further research on the antioxidant mechanism of white rot fungi under interspeicific interaction.Therefore,high-throughput sequencing and liquid chromatography-tandem mass spectrometry data identification of proteins can provide valuable resources for the analysis of related proteins involved in the antioxidant pathway in order to explore the ROS signaling pathway under the antagonistic effect of fungal mycelia.In this paper,it is first time to do a global analysis of cellular response mechanism under the interaction among three white rot fungi was performed by combining transcriptomic and proteomic data.The functions expressed by up-regulated genes in transcriptomic data were similar to the functions of highly expressed proteins in proteomic data,including antioxidant-related proteins,detoxification-related proteins and energy productionrelated proteins,it indicated that the mitochondrial respiratory system and antioxidant system in cells were significantly enhanced.Observably,the up-regulation of some genes and proteins related to nutrition competition and attack has been identified in co-cultures,including glycoside hydrolases,glucanasess,carbonate esterase family,ATPase and synthase,pathogenrelated proteins,thaumatins,chitin synthases and terpenoid synthetases,which reflected that carbohydrate and energy metabolism-related pathways were enhanced when different fungi interact with each other.At the same time,some antifungal secondary metabolites were synthesized and secreted,thereby increasing competition for territory and nutrients,and this process will also be accompanied by increased laccase production.In this paper,it was found that laccase activity of the mycelium close to the interspecific interaction zone in the three co-culture plates was also obviously higher than that in edge zone of single fungal mycelium by measuring the laccase activity of mycelium in different regions in co-culture plates,indicating that besides the physical contact of mycelium could significantly increase the laccase activity,the white rot fungi may also secreted secondary metabolites to increase laccase activity during the antagonistic interaction.In order to verify this hypothesis,extracellular metabolites secreted from mycelium in the interaction zone of the three co-cultures Tv Po,Po Ds and Tv Ds were extracted separately,and each extract was added as an inducer to the two interacting fungi of one co-culture in mono-culture.It was showed that all extracted metabolites secreted by three interaction zones could obviously induce laccase activity in three mono-cultures Tv,Po and Ds.In order to further explore and identify the key compounds related to laccase induction and find the important differentially changed metabolites in the extracellular metabolites from three co-cultures,metabolomics technology was used to analyze the extracellular metabolites under fungal co-culture and mono-culture conditions.By identifying differentially changed extracellular metabolites in the interaction zone of three cocultures,we found that the concentration and categories of secondary metabolites secreted in different co-cultures were distinct,which suggested that there were common and specific metabolic pathways in the fungal response to interspecific antagonism under interaction with different competitors.The common enhanced metabolic pathways were pyrimidine metabolism,sphingolipid metabolism,phenylalanine riboflavin metabolism and starch and sucrose metabolism pathways in the different co-cultures.The most representative specific metabolic pathway was tryptophan metabolism pathway.Unlike intracellular tryptophan which was enhanced in all co-cultues,extracellular tryptophan was only up-regulated in the co-culture of Tv Ds.In addition,most of enhanced secondary metabolites were antimicrobial compounds,such as ferulic acid,terpenoids,aromatic compounds,antibotics and mycotoxin that can inhibit protein synthesis and breakdown polysomes.Importanly,ferulic acid was most significantly enhanced in the co-cultures of Tv Po and Po Ds.Ferulic acid not only has antioxidant activity to relieve oxidative stress,but also can be used as a substrate for laccase to induce laccase activity.Therefore,the enhanced extracellular ferulic acid in three co-cultures of Tv Po,Po Ds and Tv Ds was likely to be the metabolite marker to increase laccase activity and other antioxidant enzymes activity.This paper revealed that fungal mycelium in the interaction zones of Tv Po and Po Ds could secrete a large amount of ferulic acid.Since ferulic acid has great potential application value in food,medicine,cosmetics and other fields industry,the high yield of ferulic acid obtained in the co-cultures of Tv Po and Po Ds in this research can provide a new source for the subsequent isolation and purification of ferulic acid and its application in industrial production.On the other hand,our results showed that tryptophan which was the most significantly enhanced intracellular metabolite also can significantly increase the laccase activity in three mono-cultures,and laccase induction by tryptophan was not relevant to the benzene ring structure in tryptophan.In addition,high performance liquid chromatography results showed that laccase was likely to participate in the tryptophan metabolism,thus,significant increase of laccase production under the interspecific interaction between different white rot fungi is possibly related to the enhanced intracellular tryptophan.Besides,extracellular metabolomics data showed that only co-culture of Tv Ds secreted more tryptophan compared to mono-culture,since Tv Ds secreted much less ferulic acid than Tv Po and Po Ds,so The increase of laccase activity in the interaction region of Tv Ds may also be associated with the increase of extracellular tryptophan in Tv Ds.Finally,through a conjoint analysis of intracellular and extracellular differentially changed metabolites,this paper revealeds the potential correlation between synthesis-related pathways of significantly enhanced secondary metabolites and laccase induction under interspecific interactions between white rot fungi.These metabolic pathways included glucose oxidative hydrolysis,amino acid catabolism,tricarboxylic acid cycle,terpenoid synthesis,fatty acid synthesis and catabolism,: Mitogen-activated protein kinase(MAPK)/ protein kinase A(PKA)and calcineurin(CALC)/ calmodulin(CALM)-induced signaling pathway.Based on the above conclusions,this paper reveals the antagonistic mechanism of white rot fungi at the molecular level and confirmed that the up-regulation of laccase gene and protein expression and increased laccase activity are closely related to the oxidative stress and secretion of secondary metabolites.Importantly,laccase also play an important role in the antagonistic interaction between white rot fungi.