Comparative And Evolutionary Genomics Of Sooty Blotch And Flyspeck Fungi And Their Ectophytic Mechanisms

Sooty blotch and flyspeck(SBFS)is a cosmopolitan plant fungal disease that commonly occurs in warm and humid countries and areas.It generates black smudges on the surfaces of apple,pear,bamboo,crape myrtle,etc.,leading to a substantial decline in commodity values and causing severe economic losses.This disease can be caused by a variety of fungal species,most of which belong to multiple families and genera in the class Dothideomycetes.SBFS fungi generally colonize the cuticular layers of host plants but not invade the hosts,thus considered to be a special kind of plant pathogenic fungi — "epiphytes".In recent years,systematical studies on the taxonomy,biogeography,epidemiology and chemical control of SBFS pathogens have been performed in China and abroad.However,adaptation mechanisms of the SBFS pathogens to the harsh environments of host surfaces(oligotrophy,dehydration and intense ultraviolet radiation)are still unclear.Through the whole genome sequencing,comparative genomics and transcriptomics,in combination with histological techniques,our study revealed the pathogenic mechanism and evolutionary model of SBFS pathogens.The main results are listed below:The 18.14-Mb genome of Peltaster fructicola,a widely prevalent SBFS fungus,contains a total of 8,334 protein-coding genes with a repeat content of 4.2%.Compared with other fungi,P.fructicola possesses a smaller genome size,gene number and repeat rate.The phylogenomic analysis showed that P.fructicola is closely related with the hemibiotroph Zymoseptoria tritici;however,during evolution,the former has lost more gene families(1,019 vs.525)but gained fewer gene families(58 vs.190),and the number of orphan genes in the former(1,485)is far smaller than that in the latter(2,972).Therefore,P.fructicola has undergone a reduced evolution,that is,loss of inherent genes and lack of novel genes.In silico prediction identified 107 secreted proteins in P.fructicola,which is far less than all the other selected fungi(166 to 991)including phytopathogens,saprophytes and symbionts(excluding Saccharomyces cerevisiae).And only 12 small secreted proteins(candidate effectors)were screened out in P.fructicola,which is less than all the above fungi.This indicates that P.fructicola lacks effector-mediated plant-pathogen interaction.In P.fructicola,only 15 secondary metabolite(SM)biosynthesis enzyme-coding genes were found,which is less than most of the plant pathogenic fungi(18 to 82)including Z.tritici(28),indicating that P.fructicola probably lacks secondary metabolites(e.g.,mycotoxins)that are essential to infection.Compared with the 68 plant cell wall degrading enzymes(PCWDEs)annotated in Z.tritici,P.fructicola(24)mainly lacks ?-1,4-endoglucanases,cellobiohydrolases,?-1,4-endoxylanases,?-1,4-endomannanases,pectin methyl esterases,pectate lyases and endorhamnogalacturonases.All these enzymes are involved in degradation of the backbone chains of major components(glucans,xyloglucan,xylan,galactomannan,homogalacturonan and rhamnogalacturonan I)of plant cell walls,making P.fructicola unable to break through the epidermal cell walls.In addition,the transcriptome analysis showed that most of the above three kinds of plant pathogenicity-related genes in P.fructicola were expressed either minimally or not at all during the infection process.The cutinase-coding genes were retained quite completely and have a certain level of transcriptional activity,which further proves the external pathogenicity of P.fructicola.In order to adapt to the extreme environments on host surfaces,the genes involved in the DHN-melanin(one SM)biosynthesis were also retained.Microscopic examination of the surfaces of infected apple indicated that P.fructicola can not only dissolve epicuticular waxes but also partially penetrate the cuticle proper beneath its sclerotium-like bodies.This colonization strategy undoubtedly helps P.fructicola adhere to the surfaces of host plants and acquire necessary nutrients.Therefore,we suggest that P.fructicola,a pathogen that can entirely parasitize the surfaces of host plants,should be defined as the "ectophyte",instead of the saprophytic "epiphyte".In conclusion,during evolution from a plant-penetrating pathogenic(PPP)fungus to an SBFS pathogen,P.fructicola has experienced a significant genome contraction and lost a large number of pathogenicity-related genes,but retained some functions related with the colonization on host plant surfaces,eventually leading to a unique ectophytic nutrition pattern.This evolutionary mode may have facilitated escape from host specialization and defense responses,thereby adapting to rapid environmental and ecological change.In order to prove the universality of evolutionary mode of the P.fructicola genome in the whole group of SBFS fungi,we sequenced the genomes of another three SBFS pathogens — Microcyclosporella mali,Zasmidium angulare and Microcyclospora pomicola,and then compared them with their respective PPP relatives — Mycosphaerella madeirae,Zasmidium citri and Teratosphaeria nubilosa.The comparative genome analysis showed that these three SBFS pathogens are not in competition with their PPP relatives in the genome size(26.7 Mb vs.32.2 Mb;36.2 Mb vs.42.9 Mb;22.4 Mb vs.27.1 Mb),the gene number(11,572 vs.14,017;14,946 vs.17,275;9,169 vs.10,998)and the repeat rate(1.57% vs.6.09%;3.60% vs.8.35%;1.95% vs.7.88%),indicating that their genome structures have an austerity characteristic as well.What is more,compared to their PPP relatives,all the three SBFS pathogens show contraction in the numbers of plant pathogenic factors such as secreted proteins(488 vs.633;887 vs.1,142;339 vs.484),PCWDEs(75 vs.98;140 vs.160;34 vs.75),SM biosynthesis core enzymes(29 vs.47;34 vs.49;19 vs.23)and MFS transporters(296 vs.396;511 vs.596;195 vs.242),implying their imperfect infection capacity.Therefore,we believe that the genomes of SBFS fungi all present a trend of reduced evolution;however,compared with P.fructicola,the genomes of the three SBFS fungi have a relatively smaller shrinkage degree.