Genomics Of Sooty Blotch And Flyspeck Fungi Reveals Ectophytic Evolutionary Mechanisms

Sooty blotch and flyspeck(SBFS)is worldwide fungal phytopathogens,which can form black spots and/or smudges on the surface of apples,pears and other fruits,resulting in a significant decline of their commodities and causing serious economic losses.SBFS fungi are considered to be a kind of ectophytic plant pathogenic fungi because they colonize the cuticle of plant epidermis exclusively and do not invade living cells.Previous studies have proposed the hypothesis that ectophytic pathogens originated from plant invasive-parasitic fungi and evolve from inside to outside.Our researches are carried out by using whole-genome sequencing,comparative genomics,transcriptome analysis to study: the adaptation mechanism of the SBFS fungi to the extreme micro-environment on the host surface(drought,low-nutrition,strong ultraviolet radiation,biotic stress,etc.);SBFS fungi belong to different phylogenetic branches,and whether their genomes have similar evolutionary characteristics in the process of independent evolution? Evolutionary mechanism of why SBFS fungi had small genomes.The results are as following:1.Genomic analysis of a typical SBFS fungus Ramichloridium luteum reveals that to adapt host surface lifestyle,R.luteum contracted genome contents substantially and had a drastically reduced number of pathogenic genes during evolution,such as candidate effectors,PTH11 G protein coupled receptor and plant cell wall degrading enzymes(PCWDEs).Number of PCWDEs is the least in the selected fungi,and R.luteum lost backbone gene for hydrolyzing plant cell wall components,resulting in the loss of its ability to degrade plant cell wall cellulose,hemicellulose and pectin.R.luteum has expanded more cutinase and secretory lipases related to surface cuticles.Eighteen cutinase in R.luteum is the same as Pyricularia oryzae which has the largest number in fungi kingdom.The number of secreted lipase genes was 9,far more than that of other fungi.Histological observation showed that R.luteum could efficiently degrade the cuticle.In response to drought and strong ultraviolet radiation pressures,R.luteum contain melanin synthesis genes,aquaporins and osmotic pathway related genes,and these genes were high expressed on the apple surface.Lysozyme GH25 gene can cleave peptidoglycan of bacterial cell wall and thus has antimicrobial activity.Three GH25 lysosome genes in R.luteum were the most in the selected species,and all these genes were up-regulated during surface life.These results revealed that R.luteum was to adapt ectophytic lifestyle,pathogenic genes invading plant tissues were lost,but ectophytic-pathogenic genes related to the cuticle degradation,such as cutinase and lipase,were expanded.In order to adapt to drought and strong ultraviolet radiation conditions,stress-resistant genes were retained or expanded.Therefore,we suggest that SBFS fungi enhance their ability to adapt to the surface niche through changing genome contents significantly,while avoiding the positive conflict with the host based on effective utilization of plant surface nutrition.2.SBFS fungi originated from multiple phylogenetic branches and evolved independently.In order to understand whether these fungi have similar genomic characteristics,four SBFS fungi belonging to different phylogenetic branches were selected for comparative genomic study.Genomes size of four sequenced SBFS fungi were significantly smaller than the average genome size of Ascomycetes.They contained a small number of repetitive sequences,far less than the repeats of wheat leaf blight fungi(Zymoseptoria tritici),and significantly less than the average number of Dothideomycetes species.The number of rDNA copies was very few in SBFS fungi(9 to 43),and the number of P.fructicola was reduced to minimum.Endoparasitic-pathogenic PCWDEs and candidate effectors were significantly reduced,and the most significant reduction in Schizothyrium wisconsinensis.S.wisconsinensis had six cutinase genes,and the number was the same as Peltaster fructicola.Scleroramularia shaanxiensis and Ramichloridium luteum contained 15 and 18 cutinase genes,respectively,which are expanded significantly.Secretory lipase genes(4 to 9 and up to 2 of non-SBFS fungi)were expanded in selected SBFS fungi,and the expansion degree of R.luteum was the strongest.These results indicate that different SBFS fungi evolve a large number of similar genomic characteristics during their long-term ectophytic life and similar natural environment: reducing genome size and repetitive sequence;reducing PCWDEs and candidate effector;expanding lipase genes,However,the extent of gene reduction and expansion in SBFS fungi is different,indicating that there are significant differences in the degree of evolution.3.Using Oxford Nanopore long-reads sequencing technology,we assembled the telomere-to-telomere and chromosome-scale genome of Peltaster fructicola.The final assembly size of the nuclear genome was 18.99 Mb,is the smallest yet reported for a fungus in Dothideomycetes.A total of 8072 protein-coding genes were predicted in P.fructicola and gene density was up to 418.96 per Mb,which is one of the most gene-dense or compact species in Dothideomycetes.There are very few repeats(0.3%)in P.fructicola.When compared with wheat leaf blight Z.tritici,P.fructicola reduced a large number of repeats and lost a large number of transposons families.The number of rDNA and tRNA copies are only 9 and 44,which is one of the least in fungi kingdom.Intergenic regions in P.fructicola,only occupied 31% of the genome,was the smallest proportion in Dothideomycetes.The average intron size of P.fructicola is much less than the average of Dothideomycetes species selected in this study.The number of chromosomes decreases and there are few collinear blocks between chromosomes.Most genes of P.fructicola are single copy,and one gene forms a functional cluster.Our results indicate that on the basis of expanding or retaining the essential genes for surface niche(UV,drought and microbial competition),large-scale deletion of repetitive sequences,intron length,intergenic length and redundant genes,and reducing the number of chromosomes by translocation contributed a highly compact genome structure to minimize energy and material consumption in growth and reproduction.In summary,ectophytic fungi occupy the waxy plant surface,an extreme environment characterized by prolonged desiccation,exposure to solar radiation and biotic stresses.SBFS fungi have evolved a large number of similar genomic characteristics through adjusting the changes of genome content,such as to enhance their ability to adapt to the surface niche and expand ectophytic pathogenic-related cutinase and lipase genes,while avoiding the positive conflicts with their hosts.However,the degree of evolution was varies.Finally,we believe that the low-energy is an important evolutionary dynamic for SBFS fungi to adapt nutrient stress.