Molecular Mechanism Of MiRNA Involved In The Defense Of Tea Plant Against Pseudopestalotiopsis Species

The tea plant mainly grown in the tropics and subtropics,where the warm and humid climate is conducive to the growth and reproduction of various pathogens.Therefore,there are many kinds of diseases,which cause varying degrees of losses to the yield and quality of tea.There are more than 30 kinds of tea leaf diseases recorded in China,among which the gray blight is one of the most serious diseases.Recent years,the studies are mainly focused on the identification of pathogens in local areas.However,due to the complexity of pathogens and the diversity of different areas,there are still many new species to be identified.In addition,how miRNA participate in plant disease induction and defense has become a hot topic for researchers,whereas the molecular mechanism of key miRNAs involved in tea-gray blight interaction are still not clear.Therefore,we isolated and identified the pathogens,and employed transcriptome,small RNA and degradome sequencing from 1 to 13 days post inoculation（dpi）at 3 days intervals to explore the dynamic roles of defense genes in the scenario of tea plants response to gray blight.On the one hand,it can supplement the information of new species of gray blight,on the other hand,it can reveal the molecular mechanism of miRNA participating in the defense of tea against gray blight,and provide theoretical basis for the disease control and breeding of new varieties.The main results are as follows:1.Isolation and identification of gray blightA total of 20 representative isolates were obtained from the leaves of tea plants affected by gray blight in two main tea plantations located in Anhui province,China.Further analyses showed that 18 isolates were identified as Pseudopestalotiopsis spp.,of these,14isolates were identified as new species;two isolates were identified as Neopestalotiopsis ellipsospora,on the basis of morphological characteristics and multigene phylogenetic analyses.Pathogenicity tests indicated that 4 isolates belonging to Pseudopestalotiopsis were highly aggressive isolates,whereas 2 isolates belonging to Neopestalotiopsis could not induce lesions.Furthermore,ten cultivars of tea plants were selected to be inoculated by six representative isolates involved in Pseudopestalotiopsis and Neopestalotiopsis.There were significant differences in resistance to the same isolate among tea varieties,among which"Huangshandanzhu"was the most resistant.2.The molecular mechanism of miRNAs involved in tea plants resisted to gray blightIn this study,we employed transcriptome,small RNA and degradome sequencing from 1 to 13 days post inoculation（dpi）at 3 days of tea plant under gray blight stress.A total of2116 differentially expressed genes（DEGs）were identified.Among these,pathogen-related genes were induced across all time points.DEGs related to synthesis of flavonoids,such as Csn-PAL and Csn-CHS,were intensively induced at 4 dpi;DEGs encoding disease-resistance genes were specifically reduced at 13 dpi,and may initiate effector-triggered immunity.A total of 95 miRNAs,including 72 known and 23 novel miRNAs,were differentially expressed under GB stress.Two hub genes,Csn-ERF96（ethylene responsive factor 96）and Csn-TLP（thaumatin-like protein）,which might play key roles in GB response,were validated via analyses of expression patterns and GUS assays.q RT-PCR analysis showed that there was a significant negative correlation between miRNAs and their target genes,respectively.3.Csn-miR477 targets the Csn-PAL and enhances the susceptibility of the tea plants to gray blightThe accumulation of Csn-miR477 significantly decreased in tea plants during Pseudopestalotiopsis species infection.Using miRNA and degradome data sets,the targeting of phenylalanine ammonia-lyase（PAL）by Csn-miR477 was validated by 5′RLM-RACE.GUS assay showed that the expression of Csn-PAL was post-transcriptionally regulated by Csn-miR477 and silenced by m RNA cleavage.Moreover,a negative correlation between the expression of Csn-miR477 and Csn-PAL was found in tea plants infected by pathogen.Furthermore,the transgenic lines overexpressing Csn-miR477 exhibited increased susceptibility to Pseudopestalotiopsis species,which was associated with reduced expression of At-PAL during infection.The degree of severity of the leaf lesions and the results of trypan blue staining showed that the plants overexpressing Csn-miR477 exhibited more severe damage upon pathogen infection than wild-type plants.In addition,more H₂O₂and O₂^-,higher malondialdehyde（MDA）contents and less superoxide dismutase（SOD）and peroxidase（POD）activities were detected in the transgenic plants than in the wild-type plants after inoculation with Pseudopestalotiopsis species.