The Molecular Mechanism Of Element Fe Regulating Tobacco Resistance To PVY~N

Tobacco vein necrosis disease is caused by potato virus Y necrosis strain(PVY~N)infection.PVY~N infection will reduce tobacco yield and quality,lead to losses of flue-cured value of tobacco leaves,and cause serious economic losses.In recent years,the studies on plant resistance to diseases and stresses by balancing plant growth and development processes with elements are paid more attention.Element Fe is one of the essential microelements in plant growth and metabolism,thus make the best use of element Fe to control plant virus infection becomes a novel research hotspot.Studies in our laboratory have shown that the element Fe can effectively induce the resistance of tobacco K326 to virus infection,however the functional mechanism is still unclear.In this study,the anti-PVY~N mechanism of tobacco regulated by element Fe was explored by Pac Bio SMRT full-length transcriptome sequencing and next generation RNA-seq,and the main results were as follows:1.Element Fe can prevent PVY~N infection and inhibit the accumulation of PVY~N RNA and CP in tobacco leaves.In this experiment,tobacco K326 was used as the plant material,and 3.36 mg/L of Fe2+ solution was sprayed on the leaf surface to observe its control effect on PVY~N in tobacco plants.Compared with the control group sprayed with H2 O,it was found that the symptoms of tobacco infected with PVY~N were delayed to a certain extent,and the incidence of tobacco was reduced within several days after the onset of symptoms.By investigating the disease index and calculating the control effect,the control effect of Fe element on PVY~N at 8 days post inoculation(dpi)was 24.78%.The accumulation of PVY~N RNA in tobacco leaves was detected by quantitative reverse transcription-PCR(q RT-PCR),and the accumulation of PVY~N CP in tobacco leaves was detected by Western blot assays.The results showed that element Fe could reduce the accumulation of PVY~N RNA and CP in tobacco leaves,and the accumulation difference was the largest compared with the control group at 5 dpi.2.In this study,full-length transcriptome sequence based on Pac Bio SMRT was used to annotate the Nicotiana tabacum L.cv.K326 genome under the action of Fe element and PVY~N.Equal mole of total RNA from tobacco leaves treated by Fe element and PVY~N was mixed to perform full-length transcriptome sequencing.A total of 24.52 Gb clean data was obtained by sequencing,and 300,269 CCS reads were extracted from the original data,of which FLNC sequences were 268,754.Clustering the FLNC sequences yielded 98,822 consensus reads,and 96,543 high-quality consensus reads were obtained by polish.A total of 3,106 fusion transcripts were obtained by fusion transcript prediction.Low-quality consensus reads were corrected using next generation transcriptome data and merged with high-quality consensus reads after removal of redundancy,which produced 54,211 transcript sequences.A total of 18,074 alternative splicing events occurred in transcripts,of which the maximum events were intron retention(10,333).A total of 5,109 genes were identified to have alternative polyadenylation,of which most(6284)had only one APA locus.Optimization of the gene structure revealed 25,299 gene loci,including 4,909 new loci,and 41,950 novel transcripts.The sequence structure analysis of the novel transcripts predicted a total of 20,435 SSRs,including 17,308 sequences of SSRs,of which the maximum was single-base repeat SSRs(Mono nucleotides,10,629).A total of 33,390 complete ORF sequences were predicted.A total of 3,086 lnc RNA transcripts were predicted,of which more than half(1826)were long intergenic non-coding RNAs.We obtained 11,488 transcription factors,of which the largest was b HLH transcription factor family,including 385 transcripts.The new transcript sequences obtained were searched for NR,Swissprot,GO,COG,KOG,Pfam and KEGG databases,and the functional annotations of 39,301 new transcripts were completed.3.In this study,RNA-seq technology was used to study the mechanism of element Fe regulating tobacco resistance to PVY~N from the perspective of host,and the functions of differentially expressed genes(DEGs)were analyzed by bioinformatics analysis.Annotation of the DEGs between the groups showed that the infection of PVY~N resulted in the down-regulated expression of most genes related to photosystem I reaction center subunit,cytochrome b6,chlorophyll a-b binding protein,photosystem II CP43 chlorophyll apoprotein and ATP synthase,suggesting that PVY~N infection caused severe damage to the photosynthetic system of tobacco leaves.Meanwhile,the accumulation of calcium binding protein gene,HSP,WRKY transcription factor,LRR receptor-like serine/threonine protein kinase gene,and calcium-dependent protein kinase gene was affected by PVY~N infection.Spraying Fe element could regulate varieties of genes in the electron transport chain of tobacco,such as ATP synthase subunit,cytochrome b6,PSI P700 chlorophyll-a apolipoprotein genes,photosystem II CP43 chlorophyll apoprotein,enhance the photosynthesis of tobacco,improve the ability of tobacco light energy into chemical energy and reduce the damage caused by PVY~N infection.In the early stage of virus infection,foliar application of Fe element may regulate calcium signal in tobacco cells by regulating the expression of calmodulin-like protein gene and calcium ion-binding protein gene to reduce virus damage to plants.Transcription factors,such as serine/threonine protein kinases(S/TKs),serine/threonine-protein phosphatases(PSPs),and ethylene response factors(ERFs),were also detected to be involved in the responses to regulation of Fe element and PVY~N infection.