Integrative transcriptomic, proteomic, and phosphoproteomic analysis on the defense response to reveals different expression patterns at the molecular level of durably resistant rice cultivar Mowanggu.
Frontiers in plant science
Rice blast, caused by is one of the most destructive diseases of rice ( L.) in most rice-cultivated areas worldwide. Mowanggu (MWG) is a traditional landrace rice variety in Yunnan with broad-spectrum and durable blast resistance against rice blast fungus. However, the underlying disease-resistance mechanisms remain unknown. An integrative transcriptomic, proteomic, and phosphoproteomic analysis of MWG was performed after inoculation with in this study. The transcriptomic and proteomic results revealed that MWG was moderately correlated at the transcriptional and protein levels. Differentially expressed genes and proteins were up-regulated and significantly enriched in protein phosphorylation, peroxisome, plant-pathogen interactions, phenylpropanoid metabolism and phenylalanine biosynthesis pathways. The phosphoproteomic profile and phosphorylated-protein-interaction network revealed that the altered phosphoproteins were primarily associated with reactive oxygen species (ROS), glycolysis, MAPK signaling pathways, and amino acid biosynthesis. In addition, a series of physiological and biochemical parameters, including ROS, soluble sugars, soluble protein and callus accumulation and defense-related enzyme activities, were used to validate the possible blast resistance mechanisms of MWG. The integrative transcriptomic, proteomic, and phosphoproteomic analysis revealed the different expression patterns at the molecular level of the durably resistant rice cultivar MWG after inoculation with , which provides insight into the molecular mechanisms of rice blast resistance.
10.3389/fpls.2023.1212510
Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors.
Peck S C,Nühse T S,Hess D,Iglesias A,Meins F,Boller T
The Plant cell
The perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used radioactive orthophosphate to pulse-label suspension-cultured cells of Arabidopsis in conjunction with two-dimensional gel electrophoresis and mass spectrometry to identify proteins that are phosphorylated rapidly in response to bacterial and fungal elicitors. One of these proteins, AtPhos43, and related proteins in tomato and rice, are phosphorylated within minutes after treatment with flagellin or chitin fragments. By measuring (32)P incorporation into AtPhos43 immunoprecipitated from extracts of elicitor-treated hormone and defense-response mutants, we found that phosphorylation of AtPhos43 after flagellin treatment but not chitin treatment is dependent on FLS2, a receptor-like kinase involved in flagellin perception. Induction by both elicitors is not dependent on salicylic acid or EDS1, a putative lipase involved in defense signaling.
10.1105/tpc.13.6.1467
Analysis of post-translational modification dynamics unveiled novel insights into Rice responses to MSP1.
Journal of proteomics
Magnaporthe oryzae snodprot1 homologous protein (MSP1) is known to function as a pathogen-associated molecular pattern (PAMP) and trigger PAMP-triggered immunity (PTI) in rice including induction of programmed cell death and expression of defense-related genes. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed and wild-type rice. Our analysis identified a total of 4666 PTMs-modified sites in rice leaves including 4292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, the PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides was significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses including signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, our study provides novel insights into post-translational mediated regulation of rice proteins in response to M. oryzae secreted PAMP which help in understanding the molecular mechanism of MSP1-induced signaling in rice in greater detail. SIGNIFICANCE: The research investigates the effect of overexpression of MSP1 protein in rice leaves on the phosphoproteome, acetylome, and ubiquitinome. The study found that MSP1 is involved in rice protein phosphorylation, particularly in signaling pathways, and identified a key component, PTAC16, in MSP1-induced signaling. The analysis also revealed MSP1's role in protein degradation and modification by inducing ubiquitination of the target rice proteins. The research identified potential kinases involved in the phosphorylation of rice proteins, including casein kinase II, 14-3-3 domain binding motif, β-adrenergic receptor kinase, ERK1,2 kinase substrate motif, and casein kinase I motifs. Overall, the findings provide insights into the molecular mechanisms underlying of MSP1 induced signaling in rice which may have implications for improving crop yield and quality.
10.1016/j.jprot.2023.104970
Comparative phosphoproteome analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars.
Li Yunfeng,Ye Zhijian,Nie Yanfang,Zhang Jian,Wang Guo-Liang,Wang Zhenzhong
Journal of proteomics
To explore the molecular mechanisms involved in the rice-Magnaporthe oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae, with emphasis on the initial biotrophic phase of the interaction. Phosphoproteomic profiling analysis led to the identification of 56 M. oryzae-regulated phosphoprotein spots. MALDI-TOF/TOF analysis unveiled 53 phosphoproteins belonging to 12 functional categories. Phosphorylation within 7 identified phosphoproteins was verified by mapping the phosphorylation sites by nanoLC-MS/MS. Although the phosphoproteins involved in photosynthesis, antioxidation, and protein folding showed similar changes in both compatible and incompatible interactions, differential regulation between the two interactions was documented for the phosphorylation status of defense-related proteins, signaling-related proteins, microtubule-associated proteins, energy-related enzymes, and amino acid synthesis-related proteins. Rice resistance is likely related to several rapidly and strongly triggered signal transduction cascades (e.g., Rac GTPases- and H2O2-mediated rice defense signaling) resulting in a multiple-level activation of defense responses. The six differentially expressed mRNA encoding proteins were measured by quantitative real-time PCR (qRT-PCR). This study provides useful clues for the further exploration of the sophisticated regulatory mechanisms of M. oryzae-rice interactions. BIOLOGICAL SIGNIFICANCE:Although large-scale identification of phosphorylated proteins has been carried out in rice, there is little report on the rice phosphoproteome in response to pathogens in general and in response to M. oryzae in particular. In this study, a time-course comparative phosphoproteomics was performed to analyze the changes of phosphoprotein profiles in rice leaves during early compatible and incompatible interactions with M. oryzae by using a combination of PEG prefractionation, Al(OH)3-MOAC, 2-DE, Pro-Q DPS and mass spectrometry. Fifty-six M. oryzae-regulated phosphoproteins were identified successfully and phosphorylation within 7 identified phosphoproteins was verified by mapping the phosphorylation sites by nanoLC-MS/MS. Our study provides novel insights into the functions of proteins involved in rice resistance and the molecular mechanism of rice resistance against M. oryzae. Furthermore, we propose the working model for the signaling pathways of rice defense in response to M. oryzae.
10.1016/j.jprot.2014.12.007
Identification of Msp1-Induced Signaling Components in Rice Leaves by Integrated Proteomic and Phosphoproteomic Analysis.
Gupta Ravi,Min Cheol Woo,Kim Yu-Jin,Kim Sun Tae
International journal of molecular sciences
MSP1 is a secreted protein that elicits defense responses in rice. However, the molecular mechanism of MSP1 action is largely elusive. Moreover, it is yet to be established whether MSP1 functions as a pathogen-associated molecular pattern (PAMP) or an effector. Here, we employed a TMT-based quantitative proteomic analysis of cytosolic as well as plasma membrane proteins to decipher the MSP1 induced signaling in rice. This approach led to the identification of 6691 proteins, of which 3049 were identified in the plasma membrane (PM), while 3642 were identified in the cytosolic fraction. A parallel phosphoproteome analysis led to the identification of 1906 phosphopeptides, while the integration of proteome and phosphoproteome data showed activation of proteins related to the proteolysis, jasmonic acid biosynthesis, redox metabolism, and MAP kinase signaling pathways in response to MSP1 treatment. Further, MSP1 induced phosphorylation of some of the key proteins including respiratory burst oxidase homologue-D (RBOHD), mitogen-activated protein kinase kinase kinase-1 (MEKK1), mitogen-activated protein kinase-3/6 (MPK3/6), calcium-dependent protein kinase (CDPK) and calmodulin (CaM) suggest activation of PAMP-triggered immunity (PTI) in response to MSP1 treatment. In essence, our results further support the functioning of MSP1 as a PAMP and provide an overview of the MSP1 induced signaling in rice leaves.
10.3390/ijms20174135
MoSDT1 triggers defense response through modulating phosphorylated proteins in rice.
Plant molecular biology
KEY MESSAGE:MoSDT1, a rice blast fungus transcription factor, is as an inducer to activate defense response through mainly mediating phosphorylated proteins in rice. Pathogen effector proteins play a dual role in infecting the host or triggering a defense response. Our previous research found a Magnaporthe oryzae effector, MoSDT1, which could activate the rice defense response when it was overexpressed in rice. However, we still know little about the mechanisms on how MoSDT1 in vivo or in vitro influences the resistance ability of rice. Our results showed that decreased ROS and increased lignin contents appeared along with significant upregulation of defense-related genes, raffinose synthesis gene, and phenylalanine ammonialyase gene. Moreover, we revealed that the contents of lignin were increased, which was in accordance with the upregulation of its precursor phenylalanine gene despite the fact that the glutamate-/thiamine-responsive genes were inhibited in MoSDT1 transgenic rice, and these indicated that MoSDT1 triggered the defense system of rice in vivo. Interestingly, in vitro studies, we further found that MoSDT1 induced the defense system by ROS synthesis, callose deposition, PR gene expression and SA/JA synthesis/signal genes using the purified prokaryotic expression system in rice plants. In addition, this defense response was confirmed to be activated by the zinc finger domain of MoSDT1 via prokaryotic expression of MoSDT1 truncated mutants in rice plants. To elucidate the regulative effects of MoSDT1 on protein phosphorylation in rice, phosphoproteome analysis was performed in both MoSDT1-transgenic and wild type rice. We found that MoSDT1 specifically up-regulated the expression levels of a few phosphorylated proteins, which were involved in multiple functions, such as biotic/abiotic stress and growth. In addition, the motifs in these specific proteins ranked the top among the top-five conserved motifs in the MoSDT1-transgenic rice. MoSDT1 played a crucial role in enhancing rice resistance by modulating several genes and signaling pathways.
10.1007/s11103-021-01201-4
Comparative phosphoproteomic analysis of blast resistant and susceptible rice cultivars in response to salicylic acid.
BMC plant biology
BACKGROUND:Salicylic acid (SA) is a significant signaling molecule that induces rice resistance against pathogen invasion. Protein phosphorylation carries out an important regulatory function in plant defense responses, while the global phosphoproteome changes in rice response to SA-mediated defense response has not been reported. In this study, a comparative phosphoproteomic profiling was conducted by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis, with two near-isogenic rice cultivars after SA treatment. RESULTS:Thirty-seven phosphoprotein spots were differentially expressed after SA treatment, twenty-nine of which were identified by MALDI-TOF/TOF MS, belonging to nine functional categories. Phosphoproteins involved in photosynthesis, antioxidative enzymes, molecular chaperones were similarly expressed in the two cultivars, suggesting SA might alleviate decreases in plant photosynthesis, regulate the antioxidant defense activities, thus improving basal resistance response in both cultivars. Meanwhile, phosphoproteins related to defense, carbohydrate metabolism, protein synthesis and degradation were differentially expressed, suggesting phosphorylation regulation mediated by SA may coordinate complex cellular activities in the two cultivars. Furthermore, the phosphorylation sites of four identified phosphoproteins were verified by NanoLC-MS/MS, and phosphorylated regulation of three enzymes (cinnamoyl-CoA reductase, phosphoglycerate mutase and ascorbate peroxidase) was validated by activity determination. CONCLUSIONS:Our study suggested that phosphorylation regulation mediated by SA may contribute to the different resistance response of the two cultivars. To our knowledge, this is the first report to measure rice phosphoproteomic changes in response to SA, which provides new insights into molecular mechanisms of SA-induced rice defense.
10.1186/s12870-019-2075-5