Molecular Modules LncRNA109897-JrCCR4-JrTLP1b And JrWRKY21-JrPTI5L-JrPR5L Regulate Walnut Resistance Against Anthracnose Caused By Colletotrichum Gloeosporioides

Walnut(Juglans regia L.)has a high fat content and is rich in linoleic acid,linolenic acid and tocopherol,which are good for human health.It is an important woody oil tree species and plays an important role in economic forest industry.However,walnut anthracnose was caused by the infection of Colletotrichum gloeosporioides(Penz.)Penz.and Sacc.,along with fruit gangrene,leaf scorched off,resulting in serious economic losses in walnut production.Mining walnut disease resistance genes and gene modules is of great significance for accelerating the genetic improvement of walnut disease resistance and promoting the healthy development of the walnut industry.Lnc RNA and its target genes and WRKY transcription factors play an important role in the innate immune process of plant disease resistance.In this paper,the analysis of the transcription of walnut leaf resistance to anthracnose found that the long non-coding RNA lnc RNA109897 and its target gene JrCCR4and transcription factor JrWRKY21 were significantly correlated with the resistance to walnut to anthracnose;Through molecular biology experiments such as VIGS,Y1H,Y2H,LUC,Pull down,Bi FC and EMSA,it was proved that the walnut molecular modules Lnc RNA109897-JrCCR4-JrTLP1b and JrWRKY21-JrPTI5L-JrPR5L responded to the infection of C.gloeosporioides and improved the resistance of walnut to anthracnose.The specific results are as follows:1.Molecular module Lnc RNA109897-JrCCR4-JrTLP1b regulates the resistance of walnut to C.gloeosporioides.1.1 Co-expression network analysis was carried out on the transcriptome data of walnut leaves in response to C.gloeosporioides infection.The results showed that JrCCR4(LOC108987657)encoding serine/threonine protein kinase was the target gene of lnc RNA109897(MSTRG.109897.16),and the expression levels of both were positively correlated with the infection of C.gloeosporioides(R~2=0.8165).q RT-PCR and LUC experiments demonstrated that lnc RNA109897 positively regulated and activated the expression of target gene JrCCR4.1.2 Walnut strains with silent expression and overexpression of lnc RNA109897 and JrCCR4 were obtained through VIGS experiments and transient overexpression experiments,respectively;The transgenic lines were inoculated with C.gloeosporioides,and it was found that both lnc RNA109897 and JrCCR4 could enhance the resistance of walnut leaves to C.gloeosporioides.At the same time,heterologous stable overexpression of lnc RNA109897 and JrCCR4 in Arabidopsis thaliana also increased the resistance of transgenic lines to C.gloeosporioides.1.3 The Y2H experiment was carried out with JrCCR4 protein as bait,and the results showed that JrTLP1b(LOC109012599)was a potential interacting protein of JrCCR4;the Pull down experiment verified the interaction between JrCCR4 and JrTLP1b.Disease resistance evaluation was carried out on walnut leaves that silently expressed and overexpressed JrTILP1b,as well as Arabidopsis thaliana lines with heterologous stable overexpression of JrTILP1b.The results showed that JrTILP1b can improve the host plant’s resistance to anthracnose.1.4 In order to further reveal the role of lnc RNA109897,JrCCR4 and JrTLP1b in regulating walnut resistance to C.gloeosporioides,in vitro protein degradation experiments and LUC experiments found that JrCCR4 could not only stabilize the protein activity of JrTLP1b but also activate the transcription of JrTLP1b.At the same time,JrTLP1b can induce the expression of lnc RNA109897 and activate the transcriptional activity of the lnc RNA109897 promoter.These results suggest that lnc RNA109897-JrCCR4-JrTLP1b forms a positive feedback loop to regulate walnut resistance against anthracnose.2.Molecular module JrWRKY21-JrPTI5L-JrPR5L regulates the resistance of walnut to C.gloeosporioides.2.1 Cluster analysis was performed on the WRKY transcription factor family genes in the transcriptome of walnut leaves in response to C.gloeosporioides,and q RT-PCR verification was used to identify the differentially expressed gene JrWRKY21 significantly associated with walnut resistance to C.gloeosporioides;Disease resistance analysis of walnut leaves silently expressing and overexpressing JrWRKY21 proved that JrWRKY21 could enhance the resistance of walnut leaves to C.gloeosporioides.2.2 Y2H,Pull down and Bi FC experiments showed that JrWRKY21 could interact with JrPTI5L in vivo and in vitro;at the same time,Y1H,EMSA and LUC experiments showed that JrWRKY21 could bind to the W-box element in the JrPTI5L promoter and activate the transcriptional expression of JrPTI5L.2.3 EMSA experiments showed that JrPTI5L can bind to the GCCGAC element in the JrPR5L promoter;LUC showed that JrPTI5L can activate the transcription of JrPR5L.The above results show that JrWRKY21 can indirectly activate the expression of JrPR5L gene through the JrWRKY21-JrPTI5L protein complex,thereby promoting the resistance of walnuts to C.gloeosporioides.