Powdery Mildews Are Characterized By Contracted Carbohydrate Metabolism And Diverse Effectors To Adapt To Obligate Biotrophic Lifestyle

Powdery mildew(Erysiphales)is a widespread plant disease caused by obligate biotrophic fungal pathogens involving species-specific interactions between hosts and the parasite.Although powdery mildews do not kill hosts,they are widespread pathogens that cause devastating damage to plants.Powdery mildew is an obligate biotrophic parasite which can not be cultured purely.A major limitation of molecular studies of powdery mildew is their genetic intractability.Comparison among different microbial genomes highlights the striking features shared by powdery mildews including genome size expansion and gene losses in secondary metabolism.Moreover,powdery mildews harbor a diverse superfamily of species-specific Candidate Secreted Effector Proteins(CSEPs),which are instrumental in modulating host immunity.To gain genomic insights into the underlying obligate biotrophic mechanisms,and further provide insight to the arms race between the host and powdery mildews.In this study,we sequenced the genome of a rubber tree powdery mildew(Oidium heveae)and showed the features of O.heveae genome and effectors.The comparative genomic analysis was conducted among the obligate biotrophs including powdery mildews,rusts and downy mildew,saprotrophs,hemibiotrophs and necrotrophs.We observed a genome-wide,massive contraction of multiple gene families in powdery mildews,such as enzymes in the carbohydrate metabolism pathway,in comparison with ascomycete phytopathogens,while the fatty acid metabolism pathway maintained its integrity.We also observed significant differences in terms of CSEP families between monocot and dicot powdery mildews,maybe due to different selection forces:while CSEPs in monocot mildews are likely subject to positive selection causing rapid expansion,CSEP families in dicot mildews are shrinking under strong purifying selection.The main results are summarized as following:1.Assembly and annotation of the Oidium heveae genomeWe sequenced the HO-73 isolate O.heveae genome.The total length of the O.heveae genome assembly is 62.28 Mbp.The final genome assembly contained 13,271 scaffolds and the length of N50 is 51.94 kbp.Repeat sequences were estimated to account for 49.17%of the genome.The genome was estimated to be?110.57 Mbp using K-mer.In total,97.18%of the eukaryotic core genes were mapped in the full-length by CEGMA.We generated 6,377 high-confidence protein-coding genes for O.heveae.The gene number and proteins annotation that are similar to other powdery mildews.We further computationally identified 133 coding CSEP genes in O.heveae,which is less than in Bgh,suggesting that pathogenicity-related genes for O.heveae were species-specific.2.Contraction of pathogenicity-related gene families in powdery mildewsPowdery mildew species were clustered together and far away from the other three obligate biotrophic species P.graminis f.sp.tritici,M.laricis-populina and H.arabidopsidis.Obligate biotrophic species presented contracted repertoires within CAZymes,protease,transporter and PHI classifications,indicating that the convergent evolution among obligate biotrophic species.The powdery mildew lineage showed a contraction of significant gene families,involving in carbohydrate metabolism-related functions.Powdery mildews also lose most of the pectinase genes of carbohydrate-active enzymes family.3.Pruned carbohydrate metabolism pathways in powdery mildewsWe systematically searched a conserved set of 2,646 gene families that are present in Magnaporthe oryzae,Colletotrichum higginsianum,Fusarium graminearum,Botrytis cinerea and Sclerotinia sclerotiorum,as well as in powdery mildews.We confirmed that genes enriched for energy metabolism,lipid metabolism and primary signaling pathways.We also identified 744 gene families that are absolutely absent in all powdery mildews,these are referred to as missing ascomycete pathogen core genes(MACGs).The MACGs are enriched for carbohydrate metabolism,involving in striking loss of plant pectin degradation to glycerol.Compared with the pruned carbohydrate metabolism pathways,the complete fatty acid metabolism as a nutritional source compensation.4.CSEPs are likely subject to positive selectionWe computed Ka/Ks within orthologous genes pairwise,then found strong purifying selection signature of the conserved genes in powdery mildews.We further detected the CSEPs under positive selection while most genes were under abundant purifying selection,suggesting these are the potentially rapid evolutionary pathogenicity genes.5.Distinct characterization of CSEPs between monocot powdery mildews(Oidiumheveae and Erysiphe necator)and dicot powdery mildews(Blumeria graminis f.sp.hordei and Blumeria graminis f.sp.tritici)The CSEPs are highly variation.All the CSEP family and motif of dicot powdery mildews,as well as the ribonuclease domain,are less than the monocot powdery mildews.The powdery mildews have common ancestor genes of ribonuclease.Dicot powdery mildews exhibited contractive CSEP groups likely due to strong negative selection.Monocot powdery mildews exhibited positive selective groups with a preference for small groups and shorter proteins,the groups consisting of large groups and longer proteins displayed purifying selection.The coding CSEPs genes for O.heveae were subjected to purifying selection,suggesting that a potential association with the weak and time-consuming domestication.6.Differential expression pattern of CSEPs response to incompatible interactionWe analyse the Blumeria graminis f.sp.hordei and Arabidopsis thaliana interaction time course RNA-seq.We found that a diverse expression profile of CSEPs during the interaction process,suggesting a major up-regulated expression pattern.We demonstrated the up-regulation of plant-pathogen interaction pathway result in down-regulation in 24hpi of CSEPs genes,suggesting the key of the incompatible interaction.