Functional Studies Of Pentatricopeptide Repeat Proteins FpPpr1 And FpPpr5 In Fusarium Pseudograminearum

Fusarium crown rot is an important soil-borne fungus disease in wheat.Due to the lack of disease-resistant varieties and high-efficiency chemicals,the disease has gradually become a major threaten to the wheat production in the world and expecially in China,in recent years.Fusarium pseudograminearum（Fp）is a predominant pathogen causing Fusarium crown rot in wheat,but there are few reports to explain the regulation on development and pathogenesis.Pentatricopeptide repeat protein（Ppr）is a nuclear-encoded RNA binding protein that participates in the transcription and translation of the mitochondrial genes and regulates the process of RNA maturation through directly targeting RNA sequences.Many Ppr proteins have been well-studied focusing on in plants,animals（including humans）and yeast.Nevertheless,rare studies have been conducted to uncover the function of Ppr proteins in filamentous fungi,especially plant pathogenic fungi.This research aims to elucidate the function of the Ppr proteins in F.pseudograminearum.Genome-wide analysis indicates that there are eight ortholog Ppr-encoding genes predicted in F.pseudograminearum.Bioinformatics analysis reveal that FpPprlFpPpr8 all have PPR repeat motifs containing at 35 amino acid.3D structure prediction of the Ppr protein suggests that an obvious superhelical structure exists in FpPprl,FpPpr3,FpPpr5,FpPpr6,and FpPpr7.The expression patterns of genes encoding Ppr proteins in the vegetative mycelia and the infection stage were analyzed through transcriptome sequencing.We found that FpPPR1,FpPPR3,and FpPPR5 were highly expressed in the mycelium stage,while FpPPR2,FpPPR4,FpPPR6,and FpPPR7 were highly expressed in the later stage of infection,indicating that FpPprs may function differentially during infection of wheat by F.pseudograminearum.The genes knockout of FpPPR1-FpPPR8 were conducted through homologue recombination by PEG-mediated protoplast transformation system with the application of split-marker strategy.We successfully got FpPPRl and FpPPR5 gene deletion mutants,which were named ΔFppprl and ΔFpppr5,respectively.The local isolate designated as WZ-8A as wild-type strain（WT）was used as recipient material in the transformation assays.Compared to WZ-8A and complementary strains cFppprl,the growth of colony and aerial hyphae,conidia production and morphology of length in size,and septum number as well as the germination rate in △Fppprl was all significantly reduced.Stress testing showed that △Fppprl was more sensitive to the treatment of SDS,carotene red（CR）,and the high osmotic pressure environments（NaCl and KC1）when compared with WZ-8A and cFppprl,while it showed more tolerant to H2O2 treatment.To illustrate the effect of the FpPPR1 mutation on fungi pathogenicity,inoculation of barley leaves and wheat coleoptiles were performed.Compared to WZ-8A and complementary strains cFppprl,the pathogenicity of△Fpppr1 was significantly suppressed.Additionally,the biosynthesis of DON toxin also dramatically decreased in △Fppprl.Using mitochondria staining and FpPprl-GFP signal co-localization analysis,FpPprl is predominantly located in mitochondria.Transcriptome sequencing and RIP-seq（RNA Binding Protein Immunoprecipitation Assay,RIP）methods were applied to analyze the differentially expressed genes in △Fppprl and target genes bound by FpPprl.Transcriptome data analysis showed that 1367 genes were significantly down-regulated in △Fppprl,and GO functional enrichment analysis suggested that expression levels of genes related to oxidoreductase activity were significantly affected by the deletion mutation of FpPPRl.The transcription levels of multiple oxygen reduction cytochrome P450 genes,the PKS12（PKS polyketide synthase）gene cluster,FpMAT1-1 sexual reproductive genes and heterokaryon incompatibility genes were significantly down-regulated,these findings indicates that the FpPPR1 gene may regulate oxidation-reduction process of pathogens,vegetative growth and sexual reproduction.The transcription level of mitochondrial cytochrome oxidase COX1,COX2,and COX3 genes and NADH dehydrogenase NADH1-NADH6 genes was verified by qRT-PCR When FpPPR1 was deleted,the COX1,encoding a key subunit of mitochondrial cytochrome oxidase complex,was significantly down-regulated.RIP-seq sequencing results using Flaglabeled FpPprl strains found that mitochondrial cytochrome oxidase COX1,COX2,and COX3 genes were specifically recruited by FpPprl,and COX1 was the highest one enriched.Of the high RNA levels of COX1,COX2,COX3 bound by FpPprl were quantitatively by RIP-qPCR.Thus,FpPprl may regulate of the transcription of mitochondrial cytochrome oxidase genes COX1,COX2,and COX3.To verify this,the activity of mitochondrial complex Ⅳ in wild-type and ΔFppprl was compared followed the instruction of manufacture.As expected,the activity of cytochrome oxidase in ΔFpppr1 was significantly inhibited.These findings suggested that the mutation of FpPPR1 gene may affect the transcription process of COX1,the core subunit of mitochondrial cytochrome oxidase,thus lead to functional defects of the mitochondrial respiratory chain enzyme complex.Through pull-down and tandem mass spectrometry（MS/MS）technology,we found that FpPprl may have 26 of potential interacting proteins,in which three proteins encoded by FPSE₀8624,FPSE₀6440,FPSE₀1640,respectively,interacting with both FpPprl and sexual reproduction protein FpMatl-1-3.FPSE₀6440 encodes F-type H+transport ATP enzyme d subunit,which is associated with ATP synthesis.FPSE₀1640 encodes β-tubulin,which may be involved in the growth and development of pathogens,indicating interaction between FpPpr1 and FpMat1-1-3.FpPPR5,another pentatricopeptide repeat protein-encoding gene,was also investigated in this research.Compared to WZ-8A,the ΔFpppr5 exhibited slower colony growth,rare aerial hyphae,and sporulation reduced significantly.Stress determination revealed that the deletion mutant was sensitive to SDS and CR,but tolerant to H2O2 treatment.Interestingly,under the high osmotic pressure by NaCl and KCl treatment,the defective phenotypes were restored with normal growth rate and a large amount of aerial hyphae generated.Inoculation of wheat coleoptiles and roots in pots showed that both the virulence of mutant ΔFpppr5 and the biosynthesis of DON toxin severely decreased with similar to that of ΔFppprl.The analysis of pull-down showed that FpPp5 may have 25 interacting proteins,including mitochondrial localization proteins and glutathione S-transferase functional proteins,which were predicted to be functionally related to FpPpr5.These findings demonstrated that FpPPR5 gene may regulate the cell wall integrity of F.pseudograminearum and the DON toxin biosynthesis,resulting the decrease of fungi pathogenicity in the deletion mutation of FpPPP5.In summary,both FpPPRl and FpPPR5 in the F.pseudograminearum are essential for multiple functions,such as vegetative growth,DON toxin biosynthesis process,pathogenesis and other aspects,through maintaining mitochondrial metabolic homeostasis.