Action Mechanism And Functional Gene Identification Of Tobacco Resistance To PVY Infection Regulated By Mineral Elements Iron,magnesium And Copper

Potato virus Y（PVY）is widely distributed worldwide and mainly infects Solanaceous crops,often causing huge yield and quality losses.The mineral elements iron,magnesium and copper as plant nutrients are involved in a variety of biological processes,and play a prominent role in resistance to plant pathogen infection.Our previous studies have shown that foliar spraying of different concentrations of iron,magnesium and copper can significantly inhibit the PVY infection and alleviated symptoms in field,but its mechanism is unclear.In this paper,through full-length transcriptome sequencing and second-generation transcriptome sequencing,the regulatory effects and key genes of tobacco against PVY^N infection in response to iron,magnesium and copper were studied.Functional analysis of key genes was performed through virus-induced gene silencing and transgenic techniques to explore the roles of mineral elements in plant disease resistance.These results lay an important theoretical foundation for the nutritional prevention and control of viral diseases.The main results are as follows:1.Mineral elements iron,magnesium,copper can effectively inhibit PVY^Ninfection in tobacco,and significantly reduce the symptoms of vein necrosis.Foliar spraying 3.36 mg·L^-1 EDTA-Fe,240 mg·L^-1 Mg SO₄ and 0.8 mg·L^-1 Cu SO₄ solution on K326 leaves at 72 h and 24 h before PVY^N inoculation could inhibit virus infection and delay the onset of symptoms at the early stage of PVY^N infection.The control effects at 7 dpi could reach 44.39±0.4%,40.64±0.5%and 35.29±0.3%.After treatment with Fe,Mg and Cu,the RNA accumulation of PVY^N decreased to 68%,48%and 56%and the CP protein accumulation was also significantly lower than that of the control.Fe,Mg and Cu elements can effectively inhibit viral proliferation in tobacco plants at the early stage of PVY^Ninfection,and delay the generation and development of symptoms.2.Full-length transcriptome sequencing analysis improved the transcriptome data of tobacco K326 in response to PVY^N infection treated by mineral elements iron,magnesium and copperTo accurately obtain full-length transcripts and splice variants,the Pac Bio SMRT sequencing was performed using the pooled sample from all treatments at 1,3 and 9 dpi.A total of 27.65 GB clean reads,353,258 CCS reads,316,181 full-length non-chimera（FLNC）sequences were obtained,of which 113,580 high-quality consensus sequences were obtained.The new transcript sequences were searched in NR,Swissprot,GO,COG,KOG,Pfam and KEGG databases,and the functional annotations of 39,301 new transcripts were obtained.A total of 18,074 alternative splicing（AS）events occurred in the transcripts,including 167mutually exclusive exons,10,333 intron retentions,2,086 exon skipping,1,944 variable 5’splicing sites and 3,544 variable 3’splicing sites.At the same time,6,248 genes had one alternative polyadenylation（APA）locus,2,596 genes had 2 APA loci,1,228 genes had 3 APA loci,569 genes had 4 APA loci,280 genes had 5 APA loci,and 286 genes had more than 5 APA loci.A total of 3,086 long non-coding RNAs（lnc RNAs）were identified.According to the position of lnc RNA in the reference genome,we obtained 1,836 intergenic lnc RNA（linc RNA）,74 antisense lnc RNA,77 intron lnc RNA and 1,008 sense lnc RNA.3.The main pathways of mineral elements iron,magnesium and copper regulating tobacco resistance to PVY^N were clarified,and the main common molecular pathways were photosynthesis,plant-pathogen interaction,material metabolism and transport,and signal transduction.RNA-seq was used to analyze the effects of exogenous spraying of Fe,Mg and Cu elements on PVY^N infection at 1dpi,3dpi and 9dpi on tobacco plants.The analysis results from the data of 72 libraries showed that 16,277 DEGs in Fe treatments,11,970 DEGs in Mg treatments,and 11,938 DEGs in Cu treatments were obtained.Transcriptome sequencing analysis showed that exogenous elements of iron,magnesium and copper affected the gene expression levels of tobacco.Combined with GO,KEGG enrichment analysis and WGCNA analysis,it was found that the main pathways of tobacco in response to Fe treatments mainly included plant-pathogen interaction,transcription factor regulation,photosynthesis,protein processing in endoplasmic reticulum,glutathione metabolism,lipid metabolism and biosynthesis,cell wall biosynthesis and plant hormone signal transduction.The main molecular pathways in response to magnesium treatments mainly included energy production and conversion,oxidative phosphorylation,photosynthesis,plant-pathogen interaction,endoplasmic reticulum protein processing,inorganic salt ion transport and metabolism,organic matter transport and metabolism,respiratory burst,nitrogen metabolism and Ca²⁺signal transduction.The main molecular pathways in response to copper treatments mainly included plant-pathogen interaction,defense response activation,electron transfer of photosynthesis,transcription factor regulation,transport and metabolism of inorganic ions,metabolism and transport of organic matter,lipid transport and metabolism,signal transduction and energy generation and conversion.4.The functional genes of tobacco resistance to PVY^N regulated by iron,magnesium and copper were screened,and the genes that responded to iron,magnesium and copper to inhibit PVY^N infection and promote PVY^N infection were identified.Virus-induced gene silencing（VIGS）technology was used to identify the roles of DEGs that were screened from transcriptome sequencing in response to Fe,Mg and Cu regulating tobacco resistance to PVY^N infection.The results showed that individual silencing Nb WRKY26,Nb HSP90,Nbns LTP and Nb FAD3 in response to Fe,Nt NR and Nt PPases in response to Mg,and Nb MLP423,Nb Fd and Nb PIP2 in response to Cu could inhibit PVY^N infection.On the contrary,individual silencing Nb Cab-6a in response to Fe,Nt GBE and Nt TPS in response to Mg promoted PVY^N infection.However,silencing Nt CML36 in response to Mg did not affect PVY^Ninfection.TRV-VIGS silencing verification and Ntab EXPA11-overexpressed transgenic tobacco plants showed that overexpressed expansin gene Ntab EXPA11 could significantly inhibit the infection of PVY^N in tobacco plants,which proved that Ntab EXPA11 played an important role in resistance of tobacco plants to virus infection.In summary,this paper systematically revealed the response mechanism of tobacco to exogenous nutrient elements Fe,Mg and Cu at the molecular level,and analyzed the effect and mechanism of Fe,Mg and Cu on tobacco resistance to PVY^N,which provided new ideas and methods for studying the inhibition of mineral elements on tobacco virus disease.The key genes responding to Fe,Mg and Cu to inhibit PVY^N infection were identified,and the expression of the expansin gene Ntab EXPA11 could significantly inhibit PVY infection,which provided a new strategy for tobacco molecular disease resistance breeding and integrated control of tobacco viral diseases.