Functional Analysis Of NPS Genes In Fusarium Pseudograminearum

Fusarium pseudograminearum(Fpg)is an important pathogen of wheat,reported in China in recent years,Fusarium crown rot(FCR)caused by Fpg poses serious threat to the production of wheat.There are few reports on the molecular mechanism of pathogenicity of Fpg at home and abroad,and in-depth research in this area has important theoretical value for understanding the interaction mechanism between host and pathogen,and also for the prevention and control of FCR.Non-ribosomal peptide synthetases(NPSs or NRPSs)catalyze a variety of small molecular peptides independent ribosomal pathway,which had important biological functions and is widely used in many fields.Non-ribosomal peptide synthetases widely exist in fungi,but few studies have been done on these genes in plant pathogenic fungiBy sequencing the transcriptome of roots infected with resistant wheat varieties Zhoumai 24 and susceptible varieties Guomai 301,we found that most of the non-ribosomal peptide synthetase genes in Fpg were up-regulated.So we proposed that it may be related to the pathogenicity of Fpg.This paper systematically studies the functions of NPS genes and the main findings are as followsTranscriptome sequencing data showed that 14 of 15 NPS genes in Fpg were differentially expressed.NPS2,NPS3,NPS5,NPS6,NPS9,NPS11,NPS12,NPS14 NPS16 and NPS19 were up-regulated while NPS4,NPS10 and NPS13 were down-regulated.Nine of the NPS genes were selected for expression verification through qRT-PCR.The expressions of NPS6,NPS9,NPS10,NPS11,NPS12 and NPS13 were consistent with the transcriptome data qRT-PCR approach was used to compare the expresion of these genes in spores and in wheat coleoptiles infected with spores at 18h,24h,30h,48h,3d,5d and 7d.The results showed that NPS1,NPS6,NPS9,NPS11 and NPS 12 were up-regulated,while NPS2,NPS3,NPS10 and NPS 13 were down-regulated.The differential expression level of NPS9 was the highest both in transcriptome and qRT-PCR.Therefore,we further verified the expression of NPS9.NPS9 gene could not be amplified from the cDNA liberary prepared from spores and hyphae in vitro,while in contrast,was detected from the cDNA generated from wheat tissue infected by Fpg.It indicated that the NPS9 gene plays an important role in the infection process with plant-induced expression.The NPS9 gene encodes a protein of 819 amino acids with two functional domains,A and T.The sequence alignment showed that the homology with Fusarium culmorum and Fusarium graminearum PH-1 was 97.19%and 97.07%respectively.The knock-out strategy of split-marker with homologous sequence replacement was applied to generated the NPS9 deletion mutant from the genome of Fpg wild-type WZ-8A strain through PEG-mediated protoplast transformation.Transformants with hygromycin resistance were first screened by PCR and confirmed by Southern blot analysis,that the NPS9 gene was clean knocked out.The ?nps9 mutant showed no significant difference in colony morphology,colony growth rate,various biological stress and conidial production compared with wild-type,but the morphology of mycelium tip was deformed.The virulence of ?nps9 mutant was significantly decreased in compare with the wild-type by inoculation of wounded and intact wheat coleoptiles and barley leaves DON synthesis ability of ?nps9 mutant was significantly reduced.The FpNPS9-GFP fusion construct driven by its native promoter could restore the defects of deletion mutant to wild-type.The complemented transformants containing the NPS9-GFP fusion fragment showed no fluorescence in spores,germ tubes and hyphae in vitro.After inoculation on wheat,fluorescence was observed in the germ tubes and hyphae produced by the conidial germination.It is further indicated that the NPS9 gene is plant-induced expression.Transcriptome sequencing of wild type and ?nps9 mutant revealed that up-regulated genes belong to biological process of transporter activity in ?nps9 mutant,up-regulated genes belong to biological process of ion homeostasis in ?nps9 mutant at infection wheat at 2d and 6d,TRI genes down-regulated.In wheat,when inoculated with ?nps9 mutant the biological process related to stress and defense response were significantly down-regulated compare with the wild-type,indicating the decreased virulence of NPS9 mutant.To understand functions of NPS6,NPS11 and NPS12,The smilar approach was performed to generated ?nps6,?nps11 and ?nps12 mutants,which displayed no difference with the wild type in colony morphology and growth rate,pathogenicity and DON production were reduced compared with wild type.Our findings for all of the four NPS genes with up-regulated expression pattern were affecting the pathogenicity and the DON production.In summary,the metabolites from the non-ribosomal pathway in F.pseudograminearum is essential for its infection,which promotes infectious hyphae to overcome the host defense response and promote the synthesis of DON,play an important role in the process of infection.