The Molecular Mechanism Of Arabidopsis Thaliana Secreted Peptide PIP1 To Modulate Plant Immunity

In plants, the pathogen-associated molecular patterns (PAMP)-trigerred immunity (PTI), as a major immune signaling pathway, is initiated by plasma membrane-localized pattern recognition receptors (PRRs) upon the perception of exogenous pathogen-associated molecular patterns (PAMPs). It was also indicated that plants can activate immune responses or modulate immune outputs through the generation of some endogenous molecules, such as the peptide PEP1, the immune-related hormone salicylic acid and ethylene. Endogenous secreted peptides, as new kind of plant hormone, were found to play crutial roles in the regulation of multiple plant physiological processes, including growth, development, reproduction, and resistance to biotic and abiotic stresses. However, the mechanism of the immune modulation by secreted peptides has not be investigated fundamentally.To identify putative peptide signal components invovled in plant immune modulation, we analyzed the flg22-indcued gene expression information and identified 25 candidate peptide precursor genes. Among them, At4g28460, At4g37290 and At2g23270, belong to a same unkown gene family, and are named prePIP (precursor of the PAMP-induced secreted peptide) 1,2 and 3, respectively. The prePIP family proteins represent as typical secreted peptide precursor proteins because they have signal peptides in their N-terminus and highly conserved regions in their C-terminus. Protein sequence blast showed that Arabidopsis owns at least 10 members of the gene family, and some monocot and dicot plants, such as soybean, grape, maize and rice, also contain the prePIP homologs, suggesting that the gene famliy is evolutionary conserved.To uncover the physiological roles and the funcational mechanisms of the gene family in plant immunity, the prePIP1, a representative member of the gene family was thoroughly investigated, including its transcriptional regulation, expression profile, the sub-cellular distribution, and active domain. The fluorescence microscopy imaging of transgenic plants carrying the the prePIP1 promoter drived GFP showed that prePIP1 is greatly expressed in guard cells, hydathodes and vascular tissues. And, the prePIP1 transcription was upregulated by treatments with pathogens, PAMPs and the immune-related hormone salicylic acid. The confocal microscopy imaging of tobacco leaves transiently expressing prePIP1::GFP fusion protein that the product of prePIP1 localized in the extracellular space. Proteolytic processing and mass spectrometric (MS) analyses indicated that the glutathione S-transferase-tagged signal peptide-deleted prePIPl (GST-API) could be cleaved at a site of its C-terminus by plant protease(s) present in the extracellular space. Exogenous application of the synthetic 13-aa PIP1 peptide corresponding to the C-terminal conserved region in prePIP1 could mimic the phenotype of root growth arrest of Arabidopsis overexpressing prePIPl, suggested that the synthetic peptide is likely to act as a functional mature peptide of prePIP1. Furthermore, we also found that hydroxylation of the sixth proline of PIP1 enhanced the peptide activity of root inhibitionTo further elucidate the immune-related functional mechanism of PIP 1 peptide, we then analyzed the PIP1 activation of plant immune responses. The FRKlp-LUC reporter could be promoted by co-expression of the full length of prePIPl, but not the truncated prePIP1 without the signal peptide or the C-ternimal SGPS-motif, suggested that prePIP1 may function in apoplast through its C-ternimal SGPS-motif. We also detected the activities of the synthetic PIP1 peptide with hydroxylated Pro-6 (PIP1-H6), and found that the modified peptide could activate multiple immune responses as well, including immune-relative gene expression, MAPK activation, ROS production, stomatal closure, callose deposition, and enhanced resistance against bacterial pathogen P. syingae DC3000. In addition, overexpression of prePIPl and prePIP2 enhanced Arabidopsis resistance against fungal pathogen F. oxysporum f.sp. conglutinans 699. To identify the receptor of PIP1, we used seedling growth inhibition and immune-related gene induction in response to PIP1-H6 in in the loss-of-function mutants of some XI subfamily LRR-RLK genes, and found that the effect of PIP1-H6 was impaired in both of independent rlk7 T-DNA insertion mutants. Moreover, the PIP1-H6 induced MAPK phosphorylation and pathogen resistance were also disappeared in the rlk7 mutants. Besides, pull-down, chemical cross-linking and binding assays also confirmed that RLK7 is a PIP receptor.To investigate the mechanism of signaling pathway downstream of PIP1-RLK7, we compared the signal components of PIP1-RLK7 and PEP1-PEPR1. The results showed that the PIP1-RLK7 engages overlapping and distinct immune signaling pathway with PEP1-PEPR1, and BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1) should be a joint signal component of the two signaling pathways, but BIK1 (Botrytis-induced kinase 1) probably only mediates the PEP1-PEPR1 pathway. PIP1-RLK7 and PEP1-PEPR1 cooperate in amplifying the immune responses triggered by the PAMP flg22. Furthermore, the genome-wide transcriptional analysis of Arabidopsis genes in response to PIP1 using an RNA-sequencing technique showed that PIP1-upregulated genes are greatly overlapped with the flg22 and PEP1 treatment, supporting the hypothesis that PIP1 contributes to the flg22 signaling amplification. Because the BTH-induced genes are significantly enriched in the genes commonly up-regulated by PIP1, flg22 and PEP1, so we proposed that PIP1 and PEP1 enhance the flg22 responses through activation of SA signaling. Comparison of the flg22-upregulated genes between Wt and rlk7 showed that the transcription of flg22-induced and SA signaling-evoked genes was greatly reduced in rlk7 mutants. We also found that flg22- and SA-induced PR1 transcription and DC3000 resistance were increased in prePIP1 overexpressing seedlings but were decreased in rlk7 mutants. These observations indicated that PIP1-RLK7 may amplify the flg22 signaling by activation of SA signaling pathway, and the SA signaling could also enhance the flg22-induced expression of prePIPl, suggesting the existence of the positive feedback loop between PIP1-RLK7 and SA signaling downstream of flg22-induced plant immunity.In conclusion, we identified a novel secreted peptide PIP1 together with its receptor RLK7 in Arabidopsis, and confirmed that PIP1-RLK7 and SA signaling form a positive feedback loop to amplify flg22 signaling. These studies provided significant insights into plant immune regulation by endogenous peptide signaling.