Transcriptome Analysis Of Gymnosporangium Yamadae At Different Sporulation Stages

Gymnosporangium yamadae Miyabe ex G.Yamada is a heteroecious and demicyclic rust fungus.It needs two taxonomically different plants to complete the life cycle.G.yamadae infects Juniperus chinensis L.during its telial stage and caused the globular excrescences(fungus galls)on junipers.G.yamadae overwinters in fungal galls in the form of mycelium and the teliospores extrudes from galls in early spring.Hence its teliospores do not need to be subjected to a dormancy process as the teliospores of other rust fungi on angiosperms during the winter.Then,it alternates to Malus plant during its aecial stage and causes defoliation of the tree leaves,which has a negative impact on the fruit production and quality.Gymnosporangium is the only rust group reported on gymnosperms during its telial stage,while most heteroaecious rust fungi hold angiosperms as the telial hosts.The molecular mechanism underlying the long-term evolution of G.yamadae and gymnosperms is a major problem needed to be solved considering the biology of rust fungi.Meanwhile,the transcriptional responses to the infection of G.yamadae in apple leaves during the spermogonial and aecial stages and the gene expression profile of G.yamadae during different sporulation stages were largely unknown and deserve in-depth study.High-throughput sequencing technology combined with bioinformatics were applied in this study.The comparative transcriptomics was conducted among the three Gymnosporangium species including G.yamadae;dual RNA-seq was used to analyze the molecular responses to the G.yamadae infection of apple leaves at two time points;transcriptomes of spermogonia,aecia and telia were compared with the emphasis on the gene expression pattern.The main findings were listed below:1.The transcriptome of G.yamadae teliospores was constructed and subjected to the comparative transcriptomics analysis with the teliospores of Gymnosporangium asiaticum and Gymnosporangium japonicum.Functional annotation for the three Gymnosporangium species showed a conserved gene expression profile with the top abundant cellular categories corresponding to energy,translation and signal transduction processes.Moreover,large number of secreted proteins with unknown function were predicted from the three Gymnosporangium teliospores transcriptomes.The Markov Cluster Algorithm(MCL)analysis revealed shared and specific secreted proteins at different taxonomical levels that could represent Gymnosporangium candidate pathogenesis effectors to be functionally determined.The phylogenetic relationships of G.yamadae,G.asiaticum and G.japonicum,as well as the phylogenetic relationships among Gymnosporangium,Puccinia,Melampsora,and Cronartium were studied at the genomic level.The phylogenomic tree showed that three Gymnosporangium species formed an independent branch and were closely related to Puccinia species.At the species level,G.yamadae is more closely related to G.asiaticum than G.japonicum.2.The molecular responses of apple leaves to the infection of G.yamadae at early and late infection stages,respectively.The glutathione metabolism and amino acid sugar and nucleotide sugar biosynthesis processes were significantly enriched in the inoculated apple leaves at the early infection stage when spermogonia gathered on the upper surface of apple leaves,which participated in the detoxication process and biosynthesis of plant cell walls;several processes related to the biosynthesis of secondary metabolites were significantly enriched in the inoculated apple leaves at the late infection stage when aecia formed on the lower surface of apple leaves,the most enriched pathway is termed as the flavonoid biosynthesis process.The molecular mechanisms underlying this compatible interaction at 10 and 30 days post inoculation(dpi)indicate a significant reaction from the host plant and comprise detoxication pathways at the earliest stage to protect plant from the toxicity caused by pathogens and the induction of secondary metabolism pathways at the late stage to limit the expansion of the pathogen.3.The gene expression patterns of G.yamadae at its spermogonial,aecial and telial stages were compared in this study.G.yamadae transcript profiling indicates a conserved genetic program in the three life-cycle stages,whereas the secretome prediction reveals the presence of higher proportion of secreted proteins expressed during aecial and telial stages compared to spermogonial stage,which may contribute to the heteroecism of G.yamadae;furthermore,specific proteins and secreted proteins were identified to express during the apple leave infection stage as well as the spermogonial and aecial stages,respectively.To conclude,the teliospore transcriptomes of three Gymnosporangium species including G.yamadae were used to find the conserved gene expression pattern and specifically expressed proteins when teliospores form on gymnosperm,and the comparative transcriptomics of G.yamadae on two hosts and three sporulation stages were conducted as well,which enrich the transcriptome data in the rust research field and facilitate the comparisons among rust fungi with different life cycles;meamwhile,the analysis of molecular mechanisms underlying the interaction between G.yamadae and apple leaves layes the groundwork for taking measures to control apple rust disease.