Analysis Of F-box Genes NpPP2-B10 And NpFBA1 From N.plumbaginifolia Regulating Tobacco Black Shank Resistance

Tobacco black shank is a devastating disease caused by Phytophthora parasitica var.nicotianae,resulting in leaf degradation and even death.Nicotiana plumbaginifolia is a wild species with resistance to black shank disease and is crossbred with Nicotiana tabacum,which is an excellent breeding resource for resistance to black shank disease.F-box protein is an important component of SCF(SKP1-Cullin-F-box)E3 ligases and a decisive protein for the specific recognition of substrates to be degraded,and plays a key regulatory role in the interaction between plants and pathogens.N.plumbaginifolia is the main object of this study,and the main contents are as follows :1.Screening of plant disease-resistance related genesBased on transcriptome sequencing results of N.tabacum and N.plumbaginifolia.Genes with high expression in N.plumbaginifolia and low expression in N.tabacum after infection by P.parasiticawere screened.A total of 12 genes related to plant disease resistance were screened.The expression levels of selected genes were verified after N.plumbaginifolia infection.q PCR results showed that all disease-resistance genes were significantly up-regulated at 6 hpi,but their expression levels were different in subsequent infection time.The expression levels of four Unigene.Finally,we selected C62451.graph＿c0 and c35154.graph＿c0 genes for further analysis.2.Analysis of NpPP2-B10 gene regulation of tobacco black shank resistance(1)Cloning,subcellular localization and functional analysis of NpPP2-B10 geneNpPP2-B10(c62451.graph＿c0)consists of a conserved F-box protein motif at its N-terminal and a conserved PP2(Nictaba lectin)domain at its C-terminal.The presence of two key residues Trp 118 and Trp 125 in the PP2 domain suggests that NpPP2-B10 has potential lectin activity.The expression of NpPP2-B10 was induced by P.parasitica,SA and Me JA,but inhibited by ethylene(ETH).The overexpression vector of NpPP2-B10 was constructed and transferred into the black shank susceptible variety ‘Honghuadajinyuan’.It was found that the overexpressed NpPP2-B10 transgenic line had significant advantages in seed germination rate and plant height compared with WT plants.At the same time,the overexpressed plants increased the resistance of tobacco to black shank.(2)F-box protein characterization and lectin activity validation of NpPP2-B10 proteinSKP1 gene was proved to be the joint protein of SCF complex in E3 ligase.We further screened two SKP1 family genes NpSKP1-1A and NpSKP1-21 which response to black shank disease infection in N.plumbaginifolia.The interaction between NpPP2-B10 and NpSKP1-1A gene was verified by bimolecular fluorescence complementation(Bi FC)and yeast two-hybrid assay.NpPP2-B10 protein was obtained by constructing prokaryotic expression vector and purifying the protein,and its lectin activity was verified by using erythrocytes.It was found that NpPP2-B10 still had lectin activity in the presence of F-box domain.We also detected the lectin content in leaves of NpPP2-B10 overexpressed lines and WT plants,and found that the lectin content in overexpressed lines was significantly higher than that in WT plants.These results suggest that NpPP2-B10 gene may regulate plant growth and black shank resistance through ubiquitin protease pathway and lectins.(3)Effects of NpPP2-B10 gene on expression of disease-resistance related genes and activities of disease-resistance related enzymes in tobaccoWe detected the expression levels of some potential downstream disease-resistance related genes,and found that SA response related gene NtPR1,NtCHN50,NtPAL,and disease course related gene Nt PR2 were significantly up-regulated after black shank infection.Meanwhile,enzyme activity was also detected.It was found that catalase(CAT)and peroxidase(POD)activities of overexpressed lines increased significantly after infection,and CAT was more responsive than POD.These results suggest that NpPP2-B10 may improve plant resistance through salicylic acid pathways and increase the content of CAT and POD enzymes.In conclusion,the overexpression of NpPP2-B10 gene has a positive regulation effect on the growth and development of tobacco and tobacco black shank resistance.3.Analysis of NpFBA1 gene regulation of tobacco black shank resistanceThe NpFBA1 protein(c35154.graph＿c0)contains only one unique F-box associated(FBA)domain and does not have an F-box conserved domain.Phylogenetic analysis placed this gene and other Nicotiana FBA genes on a separate branch.The expression of NpFBA1 was induced by black shank pathogen(Phytophthora parasitica var.nicotianae)infection and treatment with SA,Me JA and ETH.NpFBA1 overexpressed lines showed leaf curl at the rosette stage,and leaf curl,roundness and dwarfing at the mature stage.Anatomical analysis of the curl part showed that leaf curl might be caused by the increase of upper epidermal cells and palisade tissue density.The overexpression of NpFBA1 gene also resulted in the decrease of lignin content.Further studies revealed that overexpression of NpFBA1 significantly downregulated the expression of auxin response factors such as Nt ARF3 and the lignin synthesis genes Nt PAL,Nt C4 H,Nt CAD2,and Nt CCR1 in the leaves.In conclusion,on the one hand,the overexpression of NpFBA1 gene can lead to leaf curl;on the other hand,it may negatively regulate the resistance of NpFBA1 to tobacco black shank disease.