Comparative Transcriptomics Provide Insights Into The Pathogenic Immune Response Mechanism Of Leaf Brown Spot In Weeping Forsythia

Forsythia suspensa（Thunb.）Vahl is a deciduous shrub of Oleaceae,whose fruit is traditional Chinese medicine Forsythiae Fructus,one of 40 major Chinese medicinal materials,has the effect of clearing heat and detoxifying,reducing swelling and dispersing knots,and dispersing wind and heat,playing an important role in the fight against COVID-19.In order to meet the demand for fructus F.suspensa and help farmers get rid of poverty,the cultivation of F.suspensa has been vigorously promoted in many places.With the continuous expansion of cultivation area,disease problems also appeared,and caused the decline of fruit yield and quality.According to our previous study,there was brown spot in F.suspensa’s leaf,which was caused by Alternaria alternata.The yield and quality of fructus cannot be guaranteed due to the large differences among individuals,so it is urgent to cultivate good varieties.So far,there are few researches on the molecular breeding mechanism of F.suspensa,and the good breeding of F.suspensa is still in the exploratory stage.This study mainly started from screening resistant varieties and studied the process of streptospora infestation with F.suspensa from the cytological level.Physiological indexes of F.suspensa fructus leaves were determined from physiological and biochemical levels,using reference transcriptome sequencing technology,and transcriptome sequencing was performed on the samples of infected plants at two different periods to screen out the key genes related to disease resistance.The pathogenicity and immune response mechanism of F.suspensa leaf brown spot were analyzed from the above three levels to provide the basis for future molecular breeding.The research methods and results of this topic are as follows:1.In September 2022,the field natural disease resistance of 36 excellent F.suspensa plants in Sanmenxia F.suspensa planting base was identified.30 leaves were randomly selected for each F.suspensa plant and repeated three times.The severity of brown spot was investigated,and the disease index,incidence rate and relative resistance index of each plant were calculated.The results showed that 15 were highly resistant to brown spot,6 were moderately resistant to brown spot,7 were susceptible to brown spot,4 were moderately susceptible and 4 were highly susceptible.2.F.suspensa leaves inoculated with pathogen 0,5 and 10 days later were observed under stereomicroscope,optical microscope,scanning electron microscope and transmission electron microscope.Stereopicroscope was used to observe the development of lesions at the inoculation site of F.suspensa.On the 10th day,cells at the inoculation site of F.suspensa leaves were necrotic and the color of leaves turned to rust;accoding to the optical microscope,the upper epidermal cells of the leaves were not damaged.On the10th day,the cells in the lower epidermis of the leaves and part of the spongy tissue were dissolved;scanning electron microscopy further revealed that Streptomyces spore began to germinate on the 3rd day,and mycelium overgrew the whole leaf on the 5th and 10th day,The cuticle and cell wall of the upper epidermis of the leaf were not damaged,while the lower surface of the leaf was seriously damaged,and mycelium invaded the inside of the leaf through stomata and scratches;through transmission electron microscopy,some cells in leaves were found to dissolve and disappear in the 5th day,the shape of chloroplasts was changed,and osmiophilic particles increased significantly.On the 10th day,the cells were seriously deformed,chloroplasts were significantly reduced,organelles were dissolved to varying degrees,and obvious mycelia could be seen.3.The physiological and biochemical indexes of forsythia leaves at 0,5 and 10 days after inoculation showed that Peroxidase（POD）,Superoxide dismutase（SOD）and Ascorbate peroxidase（APX）in forsythia leaves increased continuously at 0-10 days after inoculation,while Catalase（CAT）increased significantly at5 days.Strong resistance to the infection of streptospora;Malondialdehyde（MDA）in susceptible materials increased significantly on the 5th day and maintained an upward trend on the 10th day,indicating serious leaf damage.4.A total of 12 cDNA libraries were constructed by transcriptome sequencing of forsythia forsythia leaves treated with sterile water and bacterial solution（5d,10d）,raw reads 270 654 218 were obtained from the original readings,and 5 037 differential genes（DEGs）were identified.There are 33 031 Unigenes which are annotated by comparing them with NR,COG,GO,KEGG and GOG databases.According to the statistics of the number of differentially expressed genes between the infection group and the control group,the maximum number of differentially expressed genes was 2 926 after 5 days of infection,including 1 280 up-regulated and down-regulated 1 646 genes.After the 10th day of infection,there were 2 111 differentially expressed genes,including 1 156 up-regulated and dow,n-regulated 955 genes.KEGG and GO enrichment analysis showed that these DGEs were mainly concentrated in metabolic pathways related to cell wall and photosynthesis.Then,the differentially expressed genes of WRKY gene family and NBS-LRR gene family related to disease resistance were mined,and 11 differentially expressed genes were found in WRKY gene family,9 of which were involved in MAPK pathogen interaction signaling pathway.Thirty-two differentially expressed genes were identified in the NBS-LRR gene family,and the most involved pathways were plant-pathogen interaction pathway and MAPK interaction signaling pathway.Through the analysis of the expression and regulation of genes in the pathway,it was found that the plant defense reaction was mainly conducted through flg22,elf18,BSS,H₂O₂and Ethylene mediated signaling pathways.In this study,15 highly resistant plants were selected.Morphological,anatomical,physiological and transcriptome data were used to identify the immune response mechanism of F.forsythia to A.alternaria,which causes leaf brown spot.Alternaria was found to invade leaves from stomata and injured parts.The invasion of A.alternaria led to cell wall dissolution,cell membrane destruction and chloroplast reduction.Streptomyces infection can induce defense response（DR）and oversensitivity response（HR）in forsythe leaves through flg22-like and elf18-like peptides,ethylene,H₂O₂,and bacterial secretion system（BSS）.This study provides a theoretical basis for the pathogenesis of leaf brown spot in F.forsythia.