Analysis Of Lifestyle Transiton Of Fungal Endophyte Phomopsis Liquidambari By Genome And Transcriptome Sequencing

Phomopsis sp.belong to Sphaeropsidaceae(Coelamycetes,Sphaeropsidales),are common inhabitants of natural ecosystems and,as saprophytes,are largely responsible for the destructive decay of litterfall,promoting the carbon and nitrogen cycles.Phomopsis liquidambari B3 was isolated from the inner bark of Bischofia polycarpa,can establish mutualistic symbiosis with a broad spectrum of crop plants.Endophytic P.liquidambari promotes rice growth,increasing rice yield and improving the efficiency of nitrogen fertilizer.This species's saprophytic counterpart can decompose rice litterfall,promoting litter organic matter cycling and the release of nutrients and improving the soil microbial environment.However,the mechanisms that trigger the switch from endophytic fungi to saprophytic fungi,and plant confront with the colonization of these endophytic fungi,are largely unexplored.Therefore,the interaction system of P.liquidambari and rice was used in this study,the interaction between fungus and plant was investigated using high-throughput sequencing techniques and bioinformatic methods.This study aims to find the endophytic and saprophytic features in fungal genome and transcriptome,clarify the key factors of P.liquidambari lifestyle transiton and mutualistic symbiosis with rice.Fluorescence microscopy,confocal laser scanning microscopy and quantitative PCR investigated the colonization dynamics and biomass of P.liquidambari in rice in vivo.P.liquidambari formed infection structures similar to phytopathogens with infected vascular tissues that systematically spread to acrial parts.However,different from pathogenic infection,P.liquidambari colonization exhibits space restriction and quantity restriction.Colonizing dynamics observations and a growth promotion assay of rice and Arabidopsis thaliana revealed that the B3 colonization strategy is hostadapted and resulted in different growth promotions influenced by N availability.However,the biochemical mechanisms and underlying genetics of the saprophyte transition to an endophyte are poorly understood.Broad host range P.liquidambari is established in endophytic and saprophytic systems with rice(Oryza sativa L.).Direct comparison of a fungal transcriptome under three different habitats provided a better understanding of lifestyle conversion during plant-fungi interactions.The isolated total RNA of CK(pure culture),EP(endophytic culture)and FP(saprophytic culture)was subjected to Illumina transcriptome sequencing.A total of 27,401,258 raw reads were generated and 22,700 unigenes were annotated.Functional annotation indicated that carbohydrate metabolism and biosynthesis of secondary metabolites played important roles.Here,the transcriptome features of generalist P.liquidambari and highlighted gene sets involved in the lifestyle transition from saprophytism to endophytism were reported.There were 2,522 differentially expressed genes(DEGs)between the saprophytic and endophytic lifestyles.Quantitative PCR analysis validated the DEGs of RNA-seq.Analysis of DEGs between saprophytic and endophytic lifestyles revealed that most genes from amino acids metabolism,carbohydrate metabolism,fatty acid biosynthesis,secondary metabolism and terpenoid and steroid biosynthesis were up-regulated in EP.Secondary metabolites of these pathways may affect fungal growth and development and contribute to signaling communication with the host.Most pathways of xenobiotic biodegradation and metabolism were up-regulated in FP.Cytochrome P450s play diverse vital roles in endophytism and saprophytism,as their highly specialized functions are evolutionarily adapted to various ecological niches.Most notable were genes for translation,ribosome biogenesis and MAPK signaling,several of which were only up-regulated in endophytic B3.Coordinated up-regulation of genes encoding enzymes involved in phenylalanine,tyrosine and tryptophan biosynthesis were preceded by secondary metabolite induction,which was encountered with host defense.Dissection at the molecular level facilitated a deeper understanding of P.liquidambari adaptation to hosts and the complex natural environment to play a role in sustainable agriculture and carbon and nitrogen cycles.On the basis of the genomic survey,we performed high-depth whole-genome sequencing of P.liquidambari to obtain the fine map of genome by the PacBio Sequel sequencing platform of the third-generation single-molecule sequencing technology.The total length of sequence was 56,176,230 bp,the number of Contig was 21,N50 was up to 7.8 Mb,the base coverage was 99.65%after assembly.Genomic comparative analysis of fungal species with different lifestyles revealed that P.liquidambari has common genomic characteristics in endophytic,saprophytic and pathogenic fungi.In the evolution,P.liquidambari is likely to has evolved from pathogen.The gene families of P.liquidambari have undergone significant expansion,mainly encoding carbohydrate active enzymes,cytochrome P450 enzymes,MFS superfamily transporters,etc.These specific genes may play important roles,and lead to its multiple nutritional strategies and significant differences from pathogens.In order to further understand the effects of endophytic fungus P.liquidambari colonization on the expression of host genes,we used rice plants inoculated with P.liquidambari B3 as the research material to conduct the transcriptome sequencing.The infection process of P.liquidambari was divided into three stages including the initial stage(1-3 dai),the middle stage(4-15 dai)and the late stage(>15 dai)based on the results of the systematic infection and colonization of P.liquidambari in rice.The RNA level of rice at each stage was detected and analyzed.We found that genes of stress response and endopeptidase inhibitory activity were significantly induced at the early stage through comparing the DEGs at different stages.Endophyte infection increases host secondary metabolic gene expression at a cost to energy metabolism,increases lipid metabolism gene expression,alters gene expression of immune response and growth promotion,leads to reprogramming of host metabolism.These results help to characterize the relationship between fungi and plants,the diversity of fungi for ecological adaptations and the application prospects for fungi in sustainable agriculture.