Identification And Function Analysis Of A Secretory Peptide PPI Identified From Nicotiana Benthamiana In Plant Immunity

Oomycetes represent a unique class of eukaryotic microorganisms that resemble fungi morphologically but are phylogenetic sisters of diatoms and brown algae within the Stramenopiles kingdom.Nearly all species in the oomycete genus Phytophthora are plant pathogens and some cause devastating crop diseases.P.parasitica,which has a wide host range,can cause diseases in more than 1,000 plant species,including the Tobacco Black Shank,one of the most serious diseases in tobacco production.Investigation of molecular mechanism of P.parasitica – plant interaction will be essential for breeding resistant cultivars and developing disease control strategies.Emerging evidence indicates that the endogenous Damage/Danger-Associated Molecular Patterns(DAMPs)including small secreted peptides are often induced during the compatible interaction between plants and pathogens for regulating diverse biological processes.Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and defense processes,induced generation of reactive oxygen species,defense-related gene expression,MAPK activation,callose deposition,and enhanced resistance.Through Agrobacterium tumefaciens-mediated transient expression technology,we identified a plant peptide,named as Phytophthora parasitica Induced(Nt PPI),which could enhance plant resistance against Phytophthora pathogens and PPI function as a DAMP in tobacco.The main results in this thesis are as follows.1.Nt PROPPI encodes a small secreted peptide PRECURSOR OF PEPTIDE(Nt PROPPI)containing the SGPS-Gx GH domain,and triggers immune responses in tobacco.Nt PROPPI is composed of 89 amino acid residues,including a 24-amino acid N-terminal signal peptide and a C-terminal SGPS(Ser-Gly-Pro-Ser)-Gx GH(Gly-x-Gly-His)mofit.The SGPS motif are conserved in the homologs from Arabidopsis,potato and tomato plants.Over-expressing Nt PROPPI induces immune responses on N.tabacum specifically,but not on N.benthamiana,potato(differential line R5)and tomato cultivar Alisa Craig.Five predicted proteins in N.benthamiana with an SGPS motif were identified as Nt PROPPI homologs,and two of the homologs were named as Nb PROPPI1(Niben101Scf00803g01016.1,Sequence similarity 95.6%)and Nb PROPPI2(Niben101Scf03747g00005.1,Sequence similarity 91.2%)due to their high protein sequence similarities to Nt PROPPI.2.Nb PROPPI1 and Nb PROPPI2 immune response in N.benthamiana and enhance plant resistance against P.parasitica.Their expression is induced by Phytophthora parasitica and salicylic acid(SA).Over-expressing Nb PROPPI1/2 could induce immune response on N.tabacum.The expression patterns analysis of Nb PROPPI under different stress conditions revealed that both Nb PROPPI1/2 could be induced by pathogen treatment and SA treatment,but not by ACC,Me JA or wounding.Co-silencing of Nb PROPPI1/2 by virus-induced gene silencing(VIGS)assay rendered plants more susceptible to P.parasitica.Nb PROPPI1 and Nb PROPPI2 were transient expressed in N.benthamiana leaves and followed by innoculation assay with P.parasitica.The results show that over-expression of Nb PROPPI1 and Nb PROPPI2 enhance plant resistance against P.parasitica.In addition,to confirm that Nb PROPPI1/2 can be secreted out,we transiently expressed Nb PROPPI1-GFP and Nb PROPPI2-GFP in N.benthamiana leaves using agroinfiltration,and observed the subcellular localization of these two-fused protein.The results show that obvious fluorescence signals of Nb PROPPI1-GFP and Nb PROPPI2-GFP were distributed in the periplasm after plasmolysis which proved that Nb PROPPI1/2 could be secreted to the periplasm.3.Nb PPI is a DAMP and activates plant immune responses in a BAK1 dependent manner,which C-terminal region outside of the SGPS motif in Nb PPI1 is essential for its immune function.According to the conserved C-terminal sequence of Nb PROPPI1 and Nb PROPPI2,peptides Nb PPI1 and Nb PPI2 were synthesized and analyzed for their immune activity.And the results revealed that Nb PPI1 is a DAMP which activates plant immune response in N.benthamiana: 1)Treatment of Nb PPI1 and Nb PPI2 enhanced plant resistance against P.parasitica and P.infestans.2)Treatment of Nb PPI1 induced expression of PTI related genes WRKY33 and FRK and activated mitogen-activated protein kinases(MAPKs).3)Nb PPI1 enhanced accumulation of reactive oxygen species(ROS)and callose induced by flg22.4)Treatment of Nb PPI1 inhibited root growth of N.benthamiana seedlings.In many cases,the corresponding receptor for DAMP recognition requires BAK1 for full activation.To explore whether Nb PPI1-induced immune signaling is BAK1-dependent,we generated BAK1-silenced plants using VIGS and examined the resulting immune response and disease resistance.The results showed that Nb PPI1-mediated resistance to P.parasitica was hindered in the BAK1-silenced plants.Flg22-induced ROS production was significantly decreased in BAK1-silenced plants,and the synergistic effect of Nb PPI1 on flg22-induced ROS production was almost completely abolished.Furthermore,Nb PPI1-triggered up-regulation of WRK33 was largely abolished in BAK1-silenced plants.These results suggested that Nb PPI1-induced signaling and their integration are partially dependent on BAK1.Through sequence alignment we obtained a conserved C-terminal region consisting of 8 amino acid residues that was present in Nt PROPPI,Nb PROPPI1 and Nb PROPPI2 but was absent in Arabidopsis homologs.To examine whether the conserved C-terminus is required for the immune function of Nb PPI1,a truncated peptide that contained a deletion of the 8 C-terminal amino acid residues of Nb PPI1(Nb PPI1-?C8)was synthesized and analyzed for its immune activity.Infection assays showed that the lesion sizes in leaves treated with Nb PPI1-?C8 were much larger than that treated with Nb PPI1.Root growth of seedlings treated with Nb PPI1-?C8 was no longer inhibited.The synergistic effect of-?C8 and flg22 on ROS production was abolished by the deletion of the 8 C-terminal amino acid residues of Nb PPI1.Nearly the entire immune response induced by treatment with Nb PPI1 was abolished in Nb PPI1-?C8.These results showed that the C-terminal region outside of the SGPS motif plays essential roles in Nb PPI1-mediated immune response and DAMP function.