Genome-wide Identification And Characterization Of Long Non-coding RNAs Participating In Resistance To Colletotrichum Gloeosporioides In Walnut(Juglans Regia)

Walnut(Juglans regia L.)is a diploid tree species(2n=32),with approximately 573.9Mb per 1C genome.Walnut anthracnose caused by Colletotrichum gloeosporioides(Penz.)Penz.and Sacc is currently the disastrous disease in walnut production.Long non-coding RNA(lnc RNA)plays an important role in plant disease resistance.In this study,the fruit bract of resistant individual ‘B26’(F26),the leaf of susceptible individual ‘B37’(L37),the leaf(resistant)(L423)and the fruit bract(susceptible)(F423)of ‘4-23’ individual were employed as the tested plant materials.According to the pathogenesis of C.gloeosporioides infection,it was determined that walnut fruit bracts and leaves uninfected tissue(0 hpi),the diseased tissues in the initial infection period(24 hpi),vegetative parasitism(48 hpi),saprophytic parasitism(72 hpi),and distal uninoculated tissues at 120 hpi were sampling sites and time nodes.Construct a transcriptome library of each sample,use the Illumina Hiseq 4000 platform for transcriptome sequencing,perform differential expression analysis,sequential expression analysis,and weighted gene coexpression network analysis on transcriptome data,and study the lnc RNAs and their target genes in walnut fruit bracts and leaves resistance response to C.gloeosporioides.The main findings are as follows:1.There were 22336 lnc RNAs were obtained,including 18403 unknown lnc RNAs and3933 known lnc RNAs in walnut fruit bracts.At the same time,a total of 23516 lnc RNAs were obtained in walnut leaves,including 18993 unknown lnc RNAs and 4523 known lnc RNAs.2.Analysis of lnc RNAs resistant C.gloeosporioides in walnut fruit bracts: according to the differential expression analysis,a total of 14236 differential lnc RNAs were identified in F26 and F423,including 10518 up-regulated lnc RNAs and 3718 down-regulated lnc RNAs.Compared with F423,F26 had a higher number of lnc RNAs that were differentially expressed and up-regulated in the early stage of infection(0 hpi,24 hpi).A total of 5 modules related to disease resistance were screened by WGCNA and the target genes of lnc RNAs were obtained.Bioinformatic analysis showed that the target genes of lnc RNAs were enriched in immune-related processes during the infection of C.gloeosporioides,such as activation of innate immune response,activation of immune response,jasmonic acid-mediated signaling pathway,defense response,and enriched in plant hormone signal transduction,phenylpropanoid biosynthesis and other pathways.According to the connectivity between genes and the corresponding weight values,104 hub-lnc RNAs and 132 corresponding target genes were identified in F26,including 11 potential resistant genes(R-genes),such as eukaryotic translation initiation factor 5A-2-like,receptor-like serine/threonine-protein kinase NCRK,probably inactive leucine-rich repeat receptor-like protein kinase At2g25790.3.Analysis of lnc RNAs against C.gloeosporioides in walnut leaf: according to differential expression analysis,a total of 12,493 differential lnc RNAs were identified in L423 and L37,including 6928 up-regulated lnc RNAs and 5565 down-regulated lnc RNAs.Compared with L37,L423 had a higher number of lnc RNAs differentially expressed and up-regulated in the pre-infection stage(24 hpi).A total of 5 modules related to L423 were screened by WGCNA and the target genes of lnc RNAs were obtained.Bioinformatic analysis showed that the target genes of lnc RNAs were enriched in immune-related processes during the infection of C.gloeosporioides,such as defense response,regulation of immune effects,stress response,and enriched in plant-pathogen interaction,plant hormone signal transduction and other pathways.According to the connectivity of lnc RNAs in the module and the corresponding weight values,89 hub-lnc RNAs and 110 corresponding target genes were identified in L423,including 17 potential R genes,such as probable LRR receptor-like serine/threonine-protein kinase At1g51860,serine/threonine-protein kinase-like protein CCR4,putative receptor protein kinase Zm PK1.4.Comparison of lnc RNAs related to C.gloeosporioides resistance in walnut fruit bracts and leaves: after C.gloeosporioides infection,lnc RNAs and target genes in F26 are mainly involved in immune response,innate immune response,jasmonic acid-mediated signal pathway regulation,lipid metabolism,fatty acid metabolism,pectin biosynthesis and other processes.The lnc RNAs and target genes in L423 are mainly involved in defense response,regulation of immune effect,plant pathogen interaction,flavonoid metabolism,phospholipid metabolism and other processes.5.Five hub-lnc RNAs and five target genes were selected from the fruit bracts and leaves of walnut for q RT-PCR analysis.The experimental results were consistent with the RNA-seq results,which verified the accuracy of the sequencing results.In summary,this study identified the key lnc RNA and corresponding target genes in the process of walnut resistance to C.gloeosporioides,and discussed the function of lnc RNAs.To further determine the specific functional mechanism of lnc RNAs in the process of walnut to C.gloeosporioides to provide theoretical and research basis.