Integrated Biological Experiments and Proteomic Analyses of Xylem Sap Revealed the Host Response to Tomato Spotted Wilt Orthotospovirus Infection.
International journal of molecular sciences
The plant vascular system is not only a transportation system for delivering nutrients but also a highway transport network for spreading viruses. Tomato spotted wilt orthotospovirus (TSWV) is among the most destructive viruses that cause serious losses in economically important crops worldwide. However, there is minimal information about the long-distance movements of TSWV in the host plant vascular system. In this this study, we confirm that TSWV virions are present in the xylem as observed by transmission electron microscopy (TEM). Further, a quantitative proteomic analysis based on label-free methods was conducted to reveal the uniqueness of protein expression in xylem sap during TSWV infection. Thus, this study identified and quantified 3305 proteins in two groups. Furthermore, TSWV infection induced three viral structural proteins, N, Gn and Gc, and 315 host proteins differentially expressed in xylem (163 up-regulated and 152 down-regulated). GO enrichment analysis showed up-regulated proteins significantly enriched in homeostasis, wounding, defense response, and DNA integration terms, while down-regulated proteins significantly enriched in cell wall biogenesis/xyloglucan metabolic process-related terms. KEGG enrichment analysis showed that the differentially expressed proteins (DEPs) were most strongly associated with plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction. Cluster analysis of DEPs function showed the DEPs can be categorized into cell wall metabolism-related proteins, antioxidant proteins, PCD-related proteins, host defense proteins such as receptor-like kinases (RLKs), salicylic acid binding protein (SABP), pathogenesis related proteins (PR), DNA methylation, and proteinase inhibitor (PI). Finally, parallel reaction monitoring (PRM) validated 20 DEPs, demonstrating that the protein abundances were consistent between label-free and PRM data. Finally, 11 genes were selected for RT-qPCR validation of the DEPs and label-free-based proteomic analysis concordant results. Our results contribute to existing knowledge on the complexity of host plant xylem system response to virus infection and provide a basis for further study of the mechanism underlying TSWV long-distance movement in host plant vascular system.
10.3390/ijms252010907
Mechanisms of Endoplasmic Reticulum Protein Homeostasis in Plants.
International journal of molecular sciences
Maintenance of proteome integrity is essential for cell function and survival in changing cellular and environmental conditions. The endoplasmic reticulum (ER) is the major site for the synthesis of secretory and membrane proteins. However, the accumulation of unfolded or misfolded proteins can perturb ER protein homeostasis, leading to ER stress and compromising cellular function. Eukaryotic organisms have evolved sophisticated and conserved protein quality control systems to ensure protein folding fidelity via the unfolded protein response (UPR) and to eliminate potentially harmful proteins via ER-associated degradation (ERAD) and ER-phagy. In this review, we summarize recent advances in our understanding of the mechanisms of ER protein homeostasis in plants and discuss the crosstalk between different quality control systems. Finally, we will address unanswered questions in this field.
10.3390/ijms242417599
Recent advances in glycoinformatic platforms for glycomics and glycoproteomics.
Abrahams Jodie L,Taherzadeh Ghazaleh,Jarvas Gabor,Guttman Andras,Zhou Yaoqi,Campbell Matthew P
Current opinion in structural biology
Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms combined with site-specific identification and occupancy are necessary steps in this direction. Computational platforms have continued to mature assisting researchers with the interpretation of such glycomics and glycoproteomics data sets, but frequently support dedicated workflows and users rely on the manual interpretation of data to gain insights into the glycoproteome. The growth of site-specific knowledge has also led to the implementation of machine-learning algorithms to predict glycosylation which is now being integrated into glycoproteomics pipelines. This short review describes commercial and open-access databases and software with an emphasis on those that are actively maintained and designed to support current analytical workflows.
10.1016/j.sbi.2019.11.009
Comparative transcriptome profiling of a rice line carrying Xa39 and its parents triggered by Xanthomonas oryzae pv. oryzae provides novel insights into the broad-spectrum hypersensitive response.
BMC genomics
BACKGROUND:Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease worldwide. Xa39 is a resistance (R) gene with a broad-spectrum hypersensitive response (BSHR) to Xoo. Nevertheless, the molecular mechanisms of resistance mediated by Xa39 remain unclear. In this study, the transcriptome profiling of a rice line carrying Xa39 and its parents at the early stage of Xoo infection were investigated. RESULTS:A rice introgression line H471 carrying Xa39 exhibited a typical local hypersensitive response phenotype, accompanied by programmed cell death after inoculation with the Xoo Philippines' race 9b. Transcriptome profiling of H471 and its parents at 1 and 2 days post-inoculation was performed using RNA sequencing. In total, 306 differentially expressed genes (DEGs) were identified in H471 compared with its recurrent parent Huang-Hua-Zhan after inoculation with Xoo. Among them, 121 (39.5%) genes, with functional enrichments that were related to defense response, protein amino acid phosphorylation, and apoptosis, were found to be constitutively expressed. The other 185 (60.5%) genes, with GO terms that belonged to defense response, were significantly responsive to Xoo infection in H471. Ten up-regulated and 12 down-regulated genes encoding intracellular immune receptors were identified in H471 compared with Huang-Hua-Zhan. LOC_Os11g37759, which was located in the fine-mapping region harboring Xa39, is a Xa39 candidate gene. The putative BSHR-related co-regulatory networks were constructed using 33 DEGs from four functional groups, including gibberellic acid receptors and brassinosteroid regulators, which were differentially co-expressed with LOC_Os11g37759 in infected H471. Our results indicated that there might be cross-talk between the Xa39-mediated signal transduction cascades and the GA/BR signaling pathway, and that the defense mechanism was related to diverse kinases, transcription factors, post-translational regulation, and R genes. CONCLUSIONS:The present study provides the comprehensive transcriptome profile of a rice introgression line carrying Xa39 and its parents, and identifies a set of DEGs involved in BSHR mediated by Xa39. These data provide novel insights into the regulatory networks of plant disease resistance mediated by R genes, and the identified DEGs will serve as candidates for Xa39 cloning and for further understanding the molecular mechanism of BSHR.
10.1186/s12864-015-1329-3
Rice immune regulator, OsPti1a, is specifically phosphorylated at the plasma membrane.
Matsui Hidenori,Takahashi Akira,Hirochika Hirohiko
Plant signaling & behavior
OsPti1a (Pto-interacting protein 1a) has important roles in the regulation of immune responses in rice. Phosphorylation of a conserved threonine in OsPti1a is necessary to activate defense responses; however, the regulatory mechanism of OsPti1a-mediated immune responses is still obscure. Recently, we revealed that OsPti1a forms protein complex(es) at the plasma membrane and this localization is required for its function. Here, we show that membrane-localized OsPti1a was selectively phosphorylated. Additionally, phosphorylation was not required for the localization of OsPti1a at the membrane. These results suggest that OsPti1a protein is selectively regulated by its phosphorylation after OsPti1a localizes to the plasma membrane.
10.4161/15592324.2014.991569
Ethylicin Inhibition of pv. oryzicola and .
Journal of agricultural and food chemistry
Infestation of rice with the bacterium pv. oryzicola () causes the serious disease bacterial leaf streak (BLS). We studied the effect of ethylicin, a broad-spectrum bactericide, on both and . Ethylicin increases the defensive enzyme activities and defensive genes expression of rice. Ethylicin also significantly inhibited activity compared with other commercial bactericides. The half-maximal effective concentration (EC) of ethylicin was 2.12 μg/mL. It has been shown that ethylicin can inhibit quorum sensing through the production of extracellular polysaccharides and enzymes, which disrupt the cell membrane. We used proteomic analysis to identify two oxidative phosphorylation pathway proteins (ACU12_RS13405 and ACU12_RS13355) which affected the virulence of and validated them using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicate that ethylicin can increase the defense responses of rice and control proliferation.
10.1021/acs.jafc.2c07327
A proteomic insight into the MSP1 and flg22 induced signaling in Oryza sativa leaves.
Meng Qingfeng,Gupta Ravi,Min Chul Woo,Kim Jongyun,Kramer Katharina,Wang Yiming,Park Sang-Ryeol,Finkemeier Iris,Kim Sun Tae
Journal of proteomics
Previously, we reported a novel Magnaporthe oryzae- secreted protein MSP1, which triggers cell death and pathogen-associated molecular pattern (PAMP)-triggered immune (PTI) responses in rice. To investigate the MSP1 induced defense response in rice at the protein level, we employed a label-free quantitative proteomic approach, in parallel with flg22 treatment, which is a well-known elicitor. Exogenous application of MSP1 to rice leaves induced an oxidative burst, MAPK3/6 activation, and activation of pathogenesis-related genes (DUF26, PBZ, and PR-10). MaxQuant based label free proteome analysis led to the identification of 4167 protein groups of which 433 showed significant differences in response to MSP1 and/or flg22 treatment. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins associated mainly with the stress response, post-translational modification and signaling were increased in abundance. Moreover, several peroxidases and receptor kinases were induced by both the elicitors, highlighting their involvement in MSP1 and flg22 induced signaling in rice. Taken together, the results reported here contribute to our understanding of MSP1 and flg22 triggered immune responses at the proteome level, thereby increasing our overall understanding of PTI signaling in rice. BIOLOGICAL SIGNIFICANCE: MSP1 is a M. oryzae secreted protein, which triggers defense responses in rice. Previous reports have shown that MSP1 is required for the pathogenicity of rice blast fungus, however, the exact mechanism of its action and its downstream targets in rice are currently unknown. Identification of the downstream targets is required in order to understand the MSP1 induced signaling in rice. Moreover, key proteins identified could also serve as potential candidates for the generation of disease resistance crops by modulating stress signaling pathways. Therefore, here we employed, for the first time, a label-free quantitative proteomic approach to investigate the MSP1 induced signaling in rice together with flg22. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins related to the defense response, signaling and ROS detoxification were majorly increased. Thus, as an elicitor, recombinant MSP1 proteins could be utilized to inducing broad pathogen resistance in crops by priming the local immune responses.
10.1016/j.jprot.2018.04.015
Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.
Journal of proteome research
The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.
10.1021/pr501064q
Data set from the phosphoproteomic 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
Data in brief
Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice-M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides.
10.1016/j.dib.2014.12.009
Comparative Proteomic Analysis of Isolates Identifies the Differentially Expressed Proteins with Roles in Virulence.
Journal of fungi (Basel, Switzerland)
Sheath blight of rice is a destructive disease that could be calamitous to rice cultivation. The significant objective of this study is to contemplate the proteomic analysis of the high virulent and less virulent isolate of using a quantitative LC-MS/MS-based proteomic approach to identify the differentially expressed proteins promoting higher virulence. Across several rice-growing regions in Odisha, Eastern India, 58 isolates were obtained. All the isolates varied in their pathogenicity. The isolate RS15 was found to be the most virulent and RS22 was identified as the least virulent. The PCR amplification confirmed that the RS15 and RS22 belonged to the subgroup of AG1-IA with a specific primer. The proteomic information generated has been deposited in the PRIDE database with PXD023430. The virulent isolate consisted of 48 differentially abundant proteins, out of which 27 proteins had higher abundance, while 21 proteins had lower abundance. The analyzed proteins acquired functionality in fungal development, sporulation, morphology, pathogenicity, detoxification, antifungal activity, essential metabolism and transcriptional activities, protein biosynthesis, glycolysis, phosphorylation and catalytic activities in fungi. A Quantitative Real-Time PCR (qRT-PCR) was used to validate changes in differentially expressed proteins at the mRNA level for selected genes. The abundances of proteins and transcripts were positively correlated. This study provides the role of the proteome in the pathogenicity of AG1-IA in rice and underpins the mechanism behind the pathogen's virulence in causing sheath blight disease.
10.3390/jof8040370
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).
Autophagy
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
10.1080/15548627.2020.1797280
The Redox Proteome of Thiol Proteins in the Rice Blast Fungus .
Zhang Xinrong,Zhang Zhenhua,Chen Xiao-Lin
Frontiers in microbiology
Redox modification, a post-translational modification, has been demonstrated to be significant for many physiological pathways and biological processes in both eukaryotes and prokaryotes. However, little is known about the global profile of protein redox modification in fungi. To explore the roles of redox modification in the plant pathogenic fungi, a global thiol proteome survey was performed in the model fungal pathogen . A total of 3713 redox modification sites from 1899 proteins were identified through a mix sample containing mycelia with or without oxidative stress, conidia, appressoria, and invasive hyphae of . The identified thiol-modified proteins were performed with protein domain, subcellular localization, functional classification, metabolic pathways, and protein-protein interaction network analyses, indicating that redox modification is associated with a wide range of biological and cellular functions. These results suggested that redox modification plays important roles in fungal growth, conidium formation, appressorium formation, as well as invasive growth. Interestingly, a large number of pathogenesis-related proteins were redox modification targets, suggesting the significant roles of redox modification in pathogenicity of . This work provides a global insight into the redox proteome of the pathogenic fungi, which built a groundwork and valuable resource for future studies of redox modification in fungi.
10.3389/fmicb.2021.648894