Regulation Mechanism Of MiR482b In The Interaction Between Tomato And Phytophthora Infestans

Tomato,as a horticultural crop with high economic and research value,is widely grown worldwide,but it is often affected by Phytophthora infestans during its growth and development,resulting in huge economic losses.Therefore,it has become an urgent problem to excavate the key molecules in the interaction between tomato and P.infestans,clarify their mechanism of action and make use of them,so as to cultivate varieties with higher disease resistance.mi RNA plays an important regulatory role in plant response to biological stress.In our previous studies,we found that miR482b can participate in the interaction process between tomato and P.infestans,but its regulatory mechanism remains unclear.In view of this,this study explored the function,target genes and regulatory pathways of miR482b through silencing and overexpression techniques as well as analysis of degradome and transcriptome data.In combination with bioinformatics analysis and site-directed mutagenesis experiments,the endogenous molecules silencing miR482b were found and verified.The regulatory network of miR482b in tomato resistance to P.infestans was constructed.The main research contents and results analysis are as follows:(1)The miR482b silenced plants were obtained by designing and transforming STTM482 b into tomatoes with Agrobacterium-mediated leaf disc transformation method.After infection with P.infestans,the lesion diameter,disease index,bacteria content and MDA content were significantly smaller in miR482b silenced plants.The disease resistance of miR482b silenced tomatoes was enhanced.Combined with bioinformatics analysis results and degradome data,four target genes cleaved by miR482b were obtained.All of them belonged to NBS-LRR resistance gene family.These results suggested that miR482b negatively regulated tomato resistance to P.infestans by inhibiting the expression of NBS-LRR genes.(2)The target gene of miR482b was cloned from Solanum pimpinellifolium L3708.A2316 bp CDS fragment belonging to CC-NBS-LRR subfamily was obtained,which was named Sp NBS-LRR.Phylogenetic analysis showed that it had a close genetic relationship with Arabidopsis RPS2.Agrobacterium-mediated leaf disk transformation and VIGS were used to overexpress and silence Sp NBS-LRR in tomato,respectively.The results of disease resistance analysis showed that overexpressed(silenced)Sp NBS-LRR significantly enhanced(reduced)the disease resistance of tomatoes.These results indicated that Sp NBS-LRR,as a member of NBS-LRR resistance gene family,had a positive regulation effect on tomato disease resistance.(3)A total of 746 differentially expressed genes were obtained by constructing the transcriptome library of miR482b overexpressed plants and comparing with the transcriptome library of wild-type plants.After enrichment analysis,20 GO terms and 11 KEGG pathways were obtained.Combined with the results of quantitative real-time PCR in miR482b overexpressed,miR482b target gene overexpressed and wild-type tomato plants,four metabolic pathways regulated by miR482b-NBS-LRR were obtained and validated.These results indicated that the overexpression of miR482b inhibited pathways related to reactive oxygen species production,jasmonic acid and ethylene synthesis and calcium signal transduction by silencing NBS-LRR,resulting in reduced disease resistance of tomatoes.(4)The results of bioinformatics prediction used transcriptome and mi RBase data showed that 89 lnc RNA could bind to 46 mi RNA.Among them,three lnc RNA(lnc RNA23468,lnc RNA13262 and lnc RNA01308)could serve as decoys for miR482b.the mutant of lnc RNA23468 was constructed by site-directed mutation technique to verify the predicted results.In the plants overexpressed(silenced)lnc RNA23468,the expression of miR482b was significantly decreased(increased),while the expression of miR482b target gene was the opposite,and the disease resistance of plants was significantly increased(decreased).However,in the plants overexpressed lnc RNA23468 mutant,the expression of miR482b and its target genes as well as the disease resistance had little change.Above results suggested that lnc RNA23468 could increase the expression of NBS-LRR by suppressing miR482b and improve the resistance of tomato to P.infestans.In conclusion,this study explored the function of miR482b and its target genes,obtained the metabolic pathways regulated by miR482b,found and verified an endogenous molecule,lnc RNA23468,which could inhibit miR482b.This study preliminarily revealed the regulatory mechanism of miR482b in tomato resistance to P.infestans and provided a theoretical basis for tomato resistance breeding.