Functional Characterizations Of The Hexose Transporters And Circular RNAs In Colletotrichum Higginsianum

Anthracnose of cruciferous crops caused by Colletotrichum higginsianum is an important plant fungal disease.In this paper,the functions of six hexose transporters in C.higginsianum were studied.In addition,the circular RNAs(circ RNAs)of C.higginsianum were identified from the whole genome sequence,and the regulatory role of circ RNAs from non-coding regions in the growth and development of C.higginsianum were analyzed.(1)G256 is a T-DNA insertional mutant which inserted in a gene coding hexose transporter,and then six hexose transporters Ch Hxt1-Ch Hxt6 were identified in C.higginsianum.Phylogenetic analysis showed that the Ch Hxt6 was clustered with the hexose transporters of unknown functions,suggesting that it might be a new member of the hexose transporters.Ch Hxt6 contain 12 transmembrane helix domains.The Ch Hxt4 gene was significantly up-regulated in the biotrophic infection stage,while Ch Hxt6 was significantly up-regulated both in the biotrophic stage and in the necrotrophic stage.The hexose transporters coding genes Ch Hxt1-Ch Hxt6 were separately knocked out,and there were no significant differences in colony morphology and conidial germination rates among all knock out mutants and the wild-type.The virulence ofΔChhxt4 andΔCh Hxt6 mutants was significantly reduced,and the formation rate of the secondary hyphae ofΔChhxt4 andΔCh Hxt6 decreased by 43%and 62%,respectively.The pathogenicity of single-point mutant Ch Hxt6(T169S)~m and the double-point mutant Ch Hxt6(T169S+P221L)~m decreased compared to the wild-type,whereas the pathogenicity of the other single-point mutant Ch Hxt6(L221L)~m showed no significant change compared to the wild-type.It was found that the secondary hyphae formation rate of Ch Hxt6(T169S)~m and Ch Hxt6(T169S+P221L)~m decreased by44%and 52%compared with the wild-type.These results indicate that the Thr169 site is required for the pathogenic function of Ch Hxt6.The yeast strains expressing Ch Hxt6could grow on maltose,glucose,galactose,fructose and mannose as the sole carbon source,while the yeast strains expressing Ch Hxt1-Ch Hxt5 were defective in growth one or more hexoses.Showing that Ch Hxt6 can widely participate in the transport and utilization of different monosaccharides.The growth of the yeast strains containing Ch Hxt6(T169S)~m,Ch Hxt6(P221L)~m or Ch Hxt6(T169S+P221L)~m on the medium supplied with glucose or galactose as the sole carbon sources was slightly weaker than that of yeast strains expressing the complete Ch Hxt6 gene.Growth of the yeast strains expressing Ch Hxt6(T169S+P221L)~m on mannose decreased significantly,indicating that the Thr169 and Pro221 sites in Ch Hxt6 is closely related to the sugar transport function of Ch Hxt6 protein.(2)A high-throughput sequencing of circular RNA(circ RNA-Seq)in C.higginsianum was conducted and a total number of 26,876 circ RNAs were identified from mycelia,conidia and appressoria samples.More than 70%of the circ RNAs in C.higginsianum are about 200-500 nt in length.The identified circ RNAs are evenly distributed on 12 chromosomes in C.higginsianum.A number of 18,574 circ RNAs,649 circ RNAs and 507 circ RNAs were specifically expressed in mycelia,conidia and appressoria accounting for 69.1%,2.3%and 1.9%of the total,respectively.Among them,the 304 circ RNAs were co-expressed in the three samples,accounting for 1.1%of the total.Through GO and KEGG enrichment analysis of the parental genes of differentially expressed circ RNAs,it was found that the functions of those genes were mainly related to the growth,development and metabolism of C.higginsianum.By the prediction analysis of circ RNA-micro RNA interaction network,2,660 circ RNAs were found to have homologous sequences with 13 Tre-mil R in Trichoderma reesei,and their interaction relationship was speculated,indicating that the circ RNA in C.higginsianum has the potential function of adsorbing micro RNAs.We identified 3,405and 2,980 circ RNAs from the coding region of the Ustilago maydis and Botrytis cinerea from the public RNA-Seq data.Compared with C.higginsianum,the proportion of exonic circ RNAs exceeded 90%,and the parent genes of 875 circ RNAs were homologous genes in C.higginsianum,U.maydis,and B.cinerea,and 113 genes produced similar circ RNAs,indicating that many circ RNAs in plant pathogenic fungi are conserved.Ten circ RNAs from non-coding region of C.higginsianum(Ch NCRs)were selected to knock out the non-coding region sequence Ch NCR1-Ch NCR10 of circ RNAs separately.The results showed that the pathogenicity of the knockout mutants of Ch NCR1-Ch NCR10 were not significantly different from that of Ch-1.The mycelial growth rate ofΔCh NCR1,ΔCh NCR3 andΔCh NCR7 were lower than that of Ch-1.TheΔCh NCR1,ΔCh NCR3 andΔCh NCR7 strains were inoculated on PDA containing sorbitol,Congo red and Calcofluor white,and the inhibition rates of mycelial growth of theΔCh NCR1 andΔCh NCR3 mutants on sorbitol were higher than that of the wild-type,indicating that the circ RNAs derived from Ch NCR1,Ch NCR3and Ch NCR7 were involved in the vegetative mycelial growth of C.higginsianum,and the circ RNAs from Ch NCR1 and Ch NCR3 regulated the sensitivity to osmotic stress in C.higginsianum.Therefore,circ RNAs derived from non-coding regions could participate in the regulation of the growth and development of plant pathogenic fungi and the response to environmental stress.