Reversible protein ubiquitination plays essential roles in regulating cellular processes. Although many reports have described the functions of ubiquitination in plant defense responses, few have focused on global changes in the ubiquitome. To better understand the regulatory roles of ubiquitination in rice pattern-triggered immunity (PTI), we investigated the ubiquitome of rice seedlings after treatment with two pathogen-associated molecular patterns, the fungal-derived chitin or the bacterial-derived flg22, using label-free quantitative proteomics. In chitin-treated samples, 144 and 167 lysine-ubiquitination sites in 121 and 162 proteins showed increased and decreased ubiquitination, respectively. In flg22-treated samples, 151 and 179 lysine-ubiquitination sites in 118 and 166 proteins showed increased and decreased ubiquitination, respectively. Bioinformatic analyses indicated diverse regulatory roles of these proteins. The ubiquitination levels of many proteins involved in the ubiquitination system, protein transportation, ligand recognition, membrane trafficking, and redox reactions were significantly changed in response to the elicitor treatments. Notably, the ubiquitination levels of many enzymes in the phenylpropanoid metabolic pathway were up-regulated, indicating that this pathway is tightly regulated by ubiquitination during rice PTI. Additionally, the ubiquitination levels of some key components in plant hormone signaling pathways were up- or down-regulated, suggesting that ubiquitination may fine-tune hormone pathways for defense responses. Our results demonstrated that ubiquitination, by targeting a wide range of proteins for degradation or stabilization, has a widespread role in modulating PTI in rice. The large pool of ubiquitination targets will serve as a valuable resource for understanding how the ubiquitination system regulates defense responses to pathogen attack.
Ubiquitination is an essential protein modification in eukaryotic cells, which is reversible. Deubiquitinating enzymes (DUBs) catalyze deubiquitination process to reverse ubiquitination, maintain ubiquitin homeostasis or promote protein degradation by recycling ubiquitins. In order to investigate effects of deubiquitination process in plant pathogenic fungus Magnaporthe oryzae, we generated deletion mutants of MoUBP14. Ortholog of MoUbp14 was reported to play general roles in ubiquitin-mediated protein degradation in Saccharomyces cerevisiae. The ΔMoubp14 mutant lost its pathogenicity and was severely reduced in mycelial growth, sporulation, carbon source utilization, and increased in sensitivity to distinct stresses. The mutant was blocked in penetration, which could due to defect in turgor generation. It is also blocked in invasive growth, which could due to reduction in stress tolerance and nutrient utilization. Deletion of UBP14 also led to accumulation of free polyubiquitin chains. Pulldown assay identified some proteins related to carbohydrate metabolism and stress response may putatively interact with MoUbp14, including two key rate-limiting enzymes of gluconeogenesis, MoFbp1 and MoPck1. These two proteins were degraded when the glucose was supplied to M. oryzae grown in low glucose media for a short period of time (∼12 h), and this process required MoUbp14. In summary, pleiotropic phenotypes of the deletion mutants indicated that MoUbp14 is required for different developments and pathogenicity of M. oryzae.
Summary Plant pathogenic fungi elaborate numerous detoxification strategies to suppress host reactive oxygen species (ROS), but their coordination is not well‐understood. Here, we show that Sirt5‐mediated protein desuccinylation in Magnaporthe oryzae is central to host ROS detoxification. SIRT5 encodes a desuccinylase important for virulence via adaptation to host oxidative stress. Quantitative proteomics analysis identified a large number of succinylated proteins targeted by Sirt5, most of which were mitochondrial proteins involved in oxidative phosphorylation, TCA cycle, and fatty acid oxidation. Deletion of SIRT5 resulted in hypersuccinylation of detoxification‐related enzymes, and significant reduction in NADPH : NADP + and GSH : GSSG ratios, disrupting redox balance and impeding invasive growth. Sirt5 desuccinylated thioredoxin Trx2 and glutathione peroxidase Hyr1 to activate their enzyme activity, likely by affecting proper folding. Altogether, this work demonstrates the importance of Sirt5‐mediated desuccinylation in controlling fungal process required for detoxifying host ROS during M. oryzae infection.
Lipid droplets (LDs) serve as one of the major reservoirs in conidia of Magnaporthe oryzae and are quickly utilized during appressorium formation. Here, we identified a gene, LDP1, encoding a perilipin that is important for LD formation and utilization during appressorium maturation. LDP1 is highly expressed in conidium and immature appressorium. Disruption mutants of LDP1 were significantly reduced in virulence, due to appressorial turgor reduction and difficulty in penetration. LDs were significantly reduced in the Δldp1 mutant, indicating LDP1 was required for LDs formation. LDP1 was colocalized with the LDs in conidium and immature appressorium but was gradually separated during appressorium maturation. A typical intracellular triacylglycerol lipase, TGL1-2, was clearly separated with LDs in conidium and immature appressorium but was well colocalized with LDs during appressorium maturation. The subcellular localization of TGL1-2 was affected by LDP1. These data suggested that LDP1 was bound to LDs for protecting from utilization in conidia and at the early appressorium stage but was separated from LDs for lipase entering and degradation. LDP1 was phosphorylated by CPKA at Thr96, which was essential for its localization and functions. These data indicate perilipin LDP1 can coordinate LD formation and utilization for appressorium-mediated infection of M. oryzae.
Objective:To compare the growth condition of the cervical-loop epithelia under different culture conditions,and to search for a simple and practical in vitro culture method of the cervical-loop epithelia.Method: Isolation and culture the cervical-loop epithelia,cells in the control group were cultured in RPMI-1640 medium,cells in the experimental group were cultured in DMEM/F12 medium,the medium contained the same component as control group under the same condition.Cell adherence,morphology and proliferation were observed under an inverted microscope.Cell proliferation activity was detected by MTT.Result:Under the inverted microscope,cells were adhered and expanded 24 hours after incubation in both groups.At 14 days,control group cells were apoptosis,experiment group cells were growed well.Significant difference was detected in the cervical-loop epithelia proliferation by both methods(P ﹤0.05).Conclusion:DMEM/F12 medium than in RPMI-1640 is more suitable for the attachment and proliferation of the cervical loop epithelial cells.
Citrobacter koseri (C.koseri) is a normal gut microbe but can cause severe opportunistic infections in immunocompromised individuals and neonates.Due to sudden hospital outbreaks, it has recently gained attention.The lack of adequate environmental surveillance and poor understanding of the epidemiology of its spread warrant the use of more sensitive and superior analysis methods.Next-generation sequencing is a powerful and rapidly emerging tool yet to be validated for routine molecular diagnosis and epidemiology of C. koseri infections.In this study, the next generation sequencing (NGS) was used to investigate the outbreak of 4 cases of C. koseri bloodstream infection in a Neurology setting within a hospital over three days.The blood samples were tested for bacterial culture among the patients with fever (body temperature ≥38°C) and chills.Additionally, the environmental samples were also cultured.A total of 4 patients with chills and fever within three days of admission and cases of sepsis due to C. koseri were identified through routine diagnosis.C. koseri was also found in the sealing fluid of environmental samples.NGS analysis was conducted on four bacterial samples from the patient's blood.The NGS data showed that entire paired-end reads were assembled into a 4.5 Mb genome with an average GC content 54.66%.The phylogram is based on the global pan-genome, suggesting a distinct clade in the identified samples.Phylogenetic analysis of the 16S RNA showed two distinct clusters.Cluster 1 originated from CKB211, while Cluster 2 was isolated from patients in the same ward (CKB212, CKB213, and CKB214).The core-pan evolutionary analysis indicated that CKB211 had a distant evolutionary relationship with other strains and more subtle evolutionary relationships were also analyzed.This analysis is consistent with the physical distance of these patients.It strongly indicates a likely route of infection via shared saline, which was the common operational approach.The study provides unique insights into the rare infection caused by C. koseri, utilizing NGS and phylogenetic analysis.In the present study, although both 16S RNA and the core-pan phylogenetic tree can be used for the evolutionary analysis of C. koseri, the core-pan analysis involves a greater sequence and provides a more nuanced understanding of divergence.The study suggests using core-pan for the evolutionary analysis of C. koseri.However, considering the limited sample size in this study, the applicability of this method remains to be explored.
Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)