Research On The Molecular Mechanism Of Piriformospora Indica Enhanced Root Rot Resistance In Gerbera

Gerbera hybrida,one of the top five fresh cutting flowers in the world,is a model plant for studying the development and evolution of complicated inflorescences.Gerbera cultivars often suffer losses from various diseases including root rot disease,a soil-borne fungal disease mainly caused by Phytophthora cryptogea.Nowadays,this disease becomes increasingly serious and has been considered a major problem restricting the healthy development of gerbera industry.Up to now,chemical and biological controls are still the main means to deal with the gerbera root rot disease.Piriformospora indica,an arbuscular mycorrhizal fungus(AMF)-like fungus,is a novel biocontrol agent and has been proved to have the ability of improving disease resistance in a variety of plants including gerbera.In our previous study,the physiological and biochemical mechanisms underlying the P.indica enhanced root rot resistance in gerbera have been clarified,but its molecular mechanism is still unclear.In this study,the transcriptome and s RNA profiling of gerbera roots from four gerbera groups(CK: control plants;PI: plants colonized by P.indica;PC: plants inoculated with P.cryptogea;PP: plants in PI group inoculated with P.cryptogea)were studied by using high-throughput sequencing technology.And some disease resistance related genes and miRNAs regulated by P.indica were excavated and studied to reveal the molecular mechanism of disease resistance.The main results of this study are as follows:1.The transcriptome changes in gerbera roots in response to P.indica and P.cryptogea inoculationA total of 15763 differentially expressed genes(DEGs)were identified among the four groups.In the four comparisons,30 common DEGs were identified,and most of which were stress related genes(such as LOX,CPK and ROMT),suggesting that these genes played an important role in the gerbera responses to root rot disease.GO and KEGG enrichment results showed that DEGs significantly affected by P.indica were mainly enriched in linoleic acid metabolism and lipid oxides and carbohydrates biosynthesis and metabolism.DEGs significantly affected by P.cryptogea were mainly enriched in plant hormone signal transduction and phenylpropanoid metabolic pathways,which were widely involved in defense response,signal transduction and L-phenylalanine catabolism.It was worth noting that DEGs in CK vs PI,CK vs PC and PI vs PP were also enriched in MAPK signaling pathway and co-targeted to plant defensin(PDF),indicating that PDF may be a key gene induced by P.indica to resist root rot.2.Functional research of ERF in the gerbera-P.cryptogea interactionsP.indica and P.cryptogea can significantly affect the transcription level of transcription factor genes,among which AP2/ERF accounts for the largest proportion.To further clarify the function of ERF,the tobacco leaves overexpressing Gh ERF were subjected to P.cryptogea resistance assays.After 3 days,histochemical staining was performed and the transcription levels of related defense genes(Nb PR2,Nb PR3,Nb ACO,Nb LOX and Nb PDF1.2)were detected.Results showed that the transient overexpression Gh ERF suppressed greatly the infection of P.cryptogea,and the expression levels of all defense related genes except Nb PDF1.2were up-regulated in tobacco leaves overexpressing Gh ERF.It was thus implied that Gh ERF enhanced plant resistance to P.cryptogea mainly through influencing the JA/ET signaling pathway.3.The s RNAome changes in gerbera roots in response to P.indica and P.cryptogea inoculationA total of 115 DE-miRNAs,including 30 known miRNAs and 85 novel miRNAs,were identified from the four comparisons.GO and KEGG enrichment analysis of the target genes of DE-miRNAs(DETs)revealed that of DETs from the CK vs PC and PC vs PP comparisons were significantly enriched in plant hormone signal transduction pathway.Moreover,most DETs of miR171 and miR393 family members were involved in auxin and gibberellin metabolism,respectively.After P.cryptogea infection,the expression of miR393 was up-regulated but its target gene TIR1 was down-regulated,suggesting that miR393 might negatively regulated the expression of TIR1 to enhance the gerbera resistance to root rot.The miR171-SCL module might play an important role in the P.indica enhanced gerbera root rot resistance.Besides,the DETs of the CK vs PC comparison involved a variety of transcription factors.Combined transcriptome and s RNA analysis showed that 19 DETs were also identified as DEGs.They were mainly regulated by miR156,miR397 and miR403 families,suggesting that they may play a crucial role in the gerbera responses to root rot disease and the P.indica enhanced root rot resistance.4.Identification,characterization and functional analysis of gerbera PDF genesBased on transcriptome data,9 PDF genes,including 4 class I defensins(GhPDF2.1～2.4)and 5 class II defensins(GhPDF1.1～1.5),were identified and successfully cloned.The numbers of positively charged residues in their γ core motifs varied.There were temporal and spatial differences in the expression patterns of two types of defensin genes,and they showed different expression patterns in response to P.cryptogea infection.Most class I defensins were highly expressed in PC petioles,while all class II defensins were highly expressed in roots.P.cryptogea infection down-regulated greatly the expression of GhPDFs at2 days of infection(2 dpi),but the expression levels of all GhPDFs except for GhPDF2.1 peaked at 15 dpi or 18 dpi.To investigate the functions of PDFs in response to root rot disease,tobacco leaves transiently overexpressing GhPDF1.5 and GhPDF2.4 were subjected to P.cryptogea resistance assays.Results showed that the antifungal effect of GhPDF2.4 was significantly better than that of GhPDF1.5.After inoculation with P.cryptogea,the expression levels of LOX and ACO in tobacco leaves overexpressing GhPDF2.4 were significantly up-regulated,indicating that GhPDF2.4 may function in the plant-P.cryptogea interaction by influencing the expression of JA/ET-related genes,thereby quickly and effectively activating the basic defense mechanism of gerbera.However,no obvious expression change pattern differences were found in leaves overexpressing GhPDF1.5,suggesting that its functional mechanism was different from GhPDF2.4.5.The inhibitory of procaryotic expressed PDF protein on the growth and infection of P.cryptogeaTo further study the antifungal ability of PDF,the recombinant soluble protein GhPDF2.4-His was induced and purified by prokaryotic expression system.The in vitro antifungal activity of the protein and its effect on the growth of P.cryptogea were explored.Moreover,the purified GhPDF2.4 was applied to tissue culture seedlings to verify its function.Results showed that the protein could significantly inhibit the mycelial growth and delay the formation of mycelial bulk of P.cryptogea on PDA medium.Notably,the application of in vitro GhPDF2.4significantly alleviated the symptoms in tissue cultured gerbera plantlets in the early stage of P.cryptogea infection.However,after 3 days,the protein had no significant inhibitory effect on the growth of pathogens,which may be related to the gradual decrease of protein activity.