Identification And Functional Study Of Long Non-Coding RNA Related To Fusarium Graminearum Conidiation

Fusarium graminearum is one of the main pathogens of wheat,barley,corn,and other cereal scabs.Ascospores produced by sexual reproduction are the main primary infection sources of Fusarium Head Blight(FHB).FHB,which widely occurs in wheat-producing areas,not only causes a reduction in food production,but also produces a variety of toxins in the infected wheat kernels that can harm human and animal health.Lnc RNAs play an important role in many biological processes of animals,plants,and yeast,but little is known about the research on the regulation and mechanism of lncRNAs in filamentous fungi.In this study,we predicted and identified lncRNAs that were differentially expressed in the conidiation in F.graminearum by strand-specific sequencing.Based on this,the function lncRNA FgHOA-AS and lncR6731 at different development stages of F.graminearum were explored.The main results are as follows:(1)A natural antisense RNA,FgHOA-AS,is transcribed from the opposite strand of the FgHOA gene locus.FgHOA encode a homoserine O-acetyltransferase(HOA)enzyme,which is a key enzyme involved in the methionine synthesis pathway.The expression of FgHOA-AS increased significantly during the conidiation stage,and FgHOA was significantly upregulated in the sexual stage.RACE-PCR results showed that the length of FgHOA-AS is 2555nt,and the length of FgHOA is 1961nt.The mutant strain ?FgHOA exhibited a methionine auxotrophic,with yellow colonies,reduced aerial hyphae and conidial germination rate.Exogenous addition of methionine can restore pigment production,vegetative growth and conidial germination,but the morphology of conidia and ascus is abnormal,and the ascospore discharge is reduced.The results indicated that FgHOA regulates the biosynthesis of methionine,and also affects conidia morphogenesis and sexual reproduction in F.graminearum.(2)In conidiation stage,overexpression of FgHOA-AS can inhibit the transcription of FgHOA,resulting in longer conidia,and the conidial germination rate is also significantly reduced.But there is no significant differences between the FgHOAOE strains and wild type strain PH-1.In sexual reproduction stage,overexpression of FgHOA leads to an increase in the number of perithecia production and ascospore discharged.Meanwhile,we also conducted FgHOA-AS-T strain in which a terminator was inserted to the 5' end of FgHOA-AS.The expression of FgHOA was significantly increased in FgHOA-AS-T,and the phenotype was similar to FgHOAOE strains.(3)lncR6731 is a lncRNA located on the upstream of FGSG?05042 gene locus with the same transcription direction.FGSG?05042 encodes 534 amino acids with a major facilitator superfamily(MFS)domain from sugar transporter subfamily.The full length of lncR6731 and FGSG?05042 was obtained by RACE PCR,and two transcripts of lncR6731 and FGSG?05042 were also discovered.Knockout mutants of lncR6731 and FGSG?05042 were obtained by the split-marker technique.In the asexual stage,the ?lncR6731 and ?FGSG?05042 had no significant difference compared to PH-1 in hyphal growth,conidiation,conidia morphology.During sexual development,the expression levels of lncR6731 and FGSG?05042 were significantly up-regulated.The ?lncR6731 and ?FGSG?05042 could produce normal-shaped perithecia and cirrhi,but the ascospore release was significantly decreased.In comparison with PH-1,the ability of ?lncR6731 and ?FGSG?05042 strains to infect wheat was slightly reduced,but the virulence on wheat coleoptiles was significantly reduced.The FGSG?05042 and lncR6731 deletion mutant showed remarkably increased DON production in infested wheat kernels and the expression level of most TRI family gene was up-regulated significantly in comparison with PH-1,indicating that FGSG?05042 and lncR6731 negatively controls DON production in F.graminearum by influencing the expression levels of TRI gene.In addition,?FGSG?05042 was sensitive to Na Cl while performing KCl resistance,indicating that the ?FGSG?05042 may involve in osmotic pressure pathways.Carbon and nitrogen source utilization measurement shows that the deletion of FGSG?05042enhance the ability of F.graminearum to utilize cellulose,carbohydrate and nitrogen sources.The sensitivity of FGSG?05042 deletion mutant to carbendazim and some DMIs was increased.The genes encoding 14-?-demethylase(the target of DMI fungicides)Fg CYP51A and Fg CYP51C were significantly down-regulation in?FGSG?05042.Taken together,these findings indicate that FGSG?05042 and lncR6731 plays a critical role in the regulation of sexual development,DON biosynthesis and pathogenesis in F.graminearum.(4)To further study the function of lncR6731,we obtained lncR6731 silencing and lncR6731 overexpression strains respectively.During the sexual reproduction stage,the expression level of FGSG?05042 was decreased and the ability of ascospore release was reduced in lncR6731 silencing strains.Moreover,overexpression of lncR6731 promoted the ability of ascospore release and significantly enhanced the expression level of FGSG?05042.In addition,lncR6731-T strain,in which a terminator was inserted of at the 3' end of lncR6731,also showed the reduced ascospore discharge,further indicating that lncR6731 can affect the ascospores released by regulating the expression of FGSG?05042.In this study,the lncRNA FgHOA-AS and lncR6731 in F.graminearum were identified for the first time,and the biological functions of FgHOA-AS and lncR6731 in vegetative growth,asexual and sexual reproduction were clarified.These results help us to understand the mechanism of asexual and sexual reproduction of F.graminearum,and provide a theoretical basis for the plant disease control,and will also broaden and enrich our understanding of the function and mechanism of lncRNA in plant pathogens.