Analyses Of Transcriptome And MicroRNAs In Response To Cucumber Infection By Corynespora Cassiicola

Cucumber?Cucumis sativus?is one of the main vegetables in the horticultural production of facilities in China.Cucumber target leaf spot?TLS?,which is caused by the fungus Corynespora cassiicola?C.cassiicola?,seriously endangers the production of cucumber.TLS affects a wide range of cucumbers worldwide,especially in China.At present,the mechanism of cucumber resistance to C.cassiicola is not completely clear.Studies on differentially expressed genes in cucumber induced by C.cassiicola will be helpful to understand directly the gene function initiated by cucumber in response to pathogenic infection,and reveal the complex regulation mechanism of cucumber defense response.It will also be helpful to provide theoretical basis for plant disease resistance breeding and plant disease control.In this assay,we performed comprehensive sequencing of the microRNAs?miRNAs?and transcriptome of a resistant cucumber?Jinyou 38?during C.cassiicola inoculation.The results identified key miRNAs and differentially expressed genes in response to the infection of C.cassiicola;using cucumber transient overexpression technique and TRV-induced STTM-microRNA silencing technique,we further confirmed the role of these key microRNAs and their target genes in cucumber resistance to C.cassiicola infection,and revealed that the rapid synthesis and accumulation of secondary metabolites such as lignin was one of the important mechanisms of cucumber resistance to C.cassiicola.The main results are as follows:?1?Through the identification of disease resistance of several varieties of cucumber inoculated with C.cassiicola,the disease index was used to screen out that Jinyou 38 was a high-resistance variety,while the Ludixianfeng was a susceptible variety.According to the ITS sequence PCR results,the target bands were detected in both varieties at 6 hpi.The content of lignin and reactive ROS in resistant variety were significantly higher than those in susceptible variety from 6 h to 24 h after inoculation with C.cassiicola,which could respond to the stress of pathogens more quickly.?2?There were 146 DEGs in the intersection of the two groups?0 hpi vs 6 hpi and 6 hpi vs 24 hpi?.We selected 17 candidate genes for classification,and these genes were associated with plant hormones,transcription factors,primary metabolism,Ca²⁺signaling pathways,secondary metabolism and defense genes.To further explore the gene expression pattern differences between the resistant and susceptible cucumber cultivars challenged with C.cassiicola,we selected 17 candidate genes for qRT-PCR assay.?3?In this study,we identified 34 DEMs belonging to 17 families and 7 novel miRNAs with precursors using high-throughput sequencing techniques and bioinformatics analyses.The miRNAs are primarily bound to target sites by complementary pairing,and we used genomic information and transcriptome data to predict the target genes in cucumber.According to the function of target genes,8 miRNAs which may be related to TLS resistance were screened.7novel miRNAs which may be related to disease resistance were also identified,and the target genes of these novel miRNAs are all previously identified differentially expressed genes,which are mostly related to secondary metabolism.?4?The transient expression vector was constructed by In-fusion technology,and we verified the interaction between candidate miRNAs and target genes by histochemical staining and fluorescence quantitative assays of GUS.We transiently overexpressed the candidate miRNAs and target genes in cucumber cotyledons to investigate the resistance to C.cassiicola.Transient overexpression of miR164d,miR396b,Novel-miR1 and Novel-miR7 in cucumber resulted in decreased resistance to C.cassiicola,while transient overexpression of NAC?inhibited by miR164d?,APE?inhibited by miR396b?,4CL?inhibited by Novel-miR1?and PAL?inhibited by Novel-miR7?led to enhanced resistance to C.cassiicola.In addition,overexpression of 4CL and PAL downregulated lignin synthesis,and overexpression of Novel-miR1 and Novel-miR7 also downregulated lignin synthesis,indicating that the regulation of4CL and PAL by Novel-miR1 and Novel-miR7 could affect lignin content.?5?The TRV-induced STTM-miRNA silencing vector was successfully constructed and the target miRNA was successfully silenced.Identification of disease resistance and determination of physiological indexes related to reactive oxygen species and lignin metabolism showed that silencing candidate microRNAs could improve cucumber resistance to C.cassiicola.