Project description:Papain-like cysteine proteases (PLCPs) play important roles in plant defense mechanisms. Previous work identified a set of five apoplastic PLCPs (CP1A, CP1B, CP2, XCP2 and CatB) which are crucial for the orchestration of SA-dependent defense signaling and vice versa in maize (Zea mays). One central question from these findings is which mechanism is triggered by apoplastic PLCPs to induce SA-dependent defenses. By a mass spectrometry approach we discovered a novel peptide (Zip1 = Zea mays immune signaling peptide) to be enriched in apoplastic fluid upon SA treatment. Zip1 induces PR-gene expression when applied to naїve maize leaves. Moreover, it activates apoplastic PLCPs similar as SA does, suggesting Zip1 to play an important role in SA-mediated defense signaling. In vitro studies using recombinant protein showed that CP1A and CP2, but not XCP2 and CatB, release Zip1 from its pro-peptide (PROZIP1) in vitro. Strikingly, metabolite analysis showed direct induction of SA de novo synthesis by Zip1 in maize leaves. In line with this, RNA sequencing revealed that Zip1-mediated changes in maize gene expression largely resemble SA-induced responses. Consequently, Zip1 increases maize susceptibility to the necrotrophic fungal pathogen Botrytis cinerea. In summary, this study identifies the PLCP-released peptide signal Zip1, which triggers SA signaling in maize.
Project description:Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PLCPs) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PLCPs and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three salicylic acid activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.
Project description:The extracellular space within plant leaves is called the apoplast, and functions as a key battlefield between plants and pathogens. Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these RNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside of EVs, but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside of EVs and are associated with proteins. Additional analyses of these extracellular RNAs (exRNAs) revealed that they are made up both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluids, GLYCINE-RICH RNA BINDING PROTEIN 7 (GRP7), as wells as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs.
Project description:In this work, we performed high throughput sequencing of small RNA libraries in maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis) to investigate the response mediated by miRNAs in these plants under control conditions, submergence, drought and alternated drought-submergence or submergence-drought stress. After Illumina sequencing of 8 small RNA libraries, we obtained from 16,139,354 to 46,522,229 raw reads across the libraries. Bioinformatic analysis identified 88 maize miRNAs and 76 miRNAs from other plants differentially expressed in maize and/or in teosinte in response to at least one of the treatments, and revealed that a larger set of miRNAs were regulated in maize than in teosinte in response to submergence and drought stress.
Project description:Many genes involve in pathogenicity and virulence are induced only in plant or in the presence of host components. Plant apoplast is the primary site of infection for P. syringae, which obtain nutrients directly from apoplastic fluid of host plants. In this work we investigated the effect of apoplastic fluid on the transcriptomic profile of the bacterium, when grown at low temperature in minimal medium with or without apoplastic fluid extracted from healthy bean leaves.
Project description:In this study, a small RNA library from maize seed 24 hours after imbibition was sequenced by the Solexa technology. A total of 11,338,273 reads were obtained. 1,047,447 total reads representing 431 unique sRNAs matched to known maize miRNAs. Further analysis confirmed the authenticity of 115 known miRNAs belonging to 24 miRNA families and the discovery of 167 novel miRNAs in maize. Both the known and the novel miRNAs were confirmed by sequencing of a second small RNA library constructed the same way as the one used in the first sequencing. We also found 10 miRNAs that had not been reported in maize, but had been reported in other plant species. All novel sequences had not been earlier described in other plant species. In addition, seven miRNA* sequences were also obtained. Putative targets for 106 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in maize imbibed seed. This study led to the confirmation of the authenticity of 115 known miRNAs and the discovery of 167 novel miRNAs in maize. Identification of novel miRNAs resulted in significant enrichment of the repertoire of maize miRNAs and provided insights into miRNA regulation of genes expressed in imbibed seed.
Project description:Genome-wide and organ-specific landscapes of epigenetic modifications and their relationships to mRNA and smRNA transcriptomes in maize We report an integrated genome-wide analysis of DNA methylation, histone modifications, smRNAs and mRNA transcriptional activity, using maize as a model. We surveyed the epigenomes of the maize inbred line B73 in shoot and root tissue by Illumina/Solexa 1G parallel sequencing after digesting genomic DNA with a methylation-sensitive restriction enzyme and after conducting chromatin immunoprecipitations (ChIP) using antibodies that target specific histone modifications (H3K4me3, H3K9ac, H3K27me3, H3K36me3, respectively). Additionally, we profiled RNA pools (micro RNA (miRNA), siRNA and mRNA) using the same sequencing strategy. Keywords: Epigenetics, mRNA transcription and small RNAs
Project description:Intervention type:DRUG. Intervention1:Huaier, Dose form:GRANULES, Route of administration:ORAL, intended dose regimen:20 to 60/day by either bulk or split for 3 months to extended term if necessary. Control intervention1:None.
Primary outcome(s): For mRNA libraries, focus on mRNA studies. Data analysis includes sequencing data processing and basic sequencing data quality control, prediction of new transcripts, differential expression analysis of genes. Gene Ontology (GO) and the KEGG pathway database are used for annotation and enrichment analysis of up-regulated genes and down-regulated genes.
For small RNA libraries, data analysis includes sequencing data process and sequencing data process QC, small RNA distribution across the genome, rRNA, tRNA, alignment with snRNA and snoRNA, construction of known miRNA expression pattern, prediction New miRNA and Study of their secondary structure Based on the expression pattern of miRNA, we perform not only GO / KEGG annotation and enrichment, but also different expression analysis.. Timepoint:RNA sequencing of 240 blood samples of 80 cases and its analysis, scheduled from June 30, 2022..
Project description:The extracellular space within plant leaves is called the apoplast, and functions as a key battlefield between plants and pathogens. Previously, we have shown that apoplastic wash fluid purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these RNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside of EVs, but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside of EVs and are associated with proteins. Additional analyses of these extracellular RNAs (exRNAs) revealed that they are made up both sRNAs and long non-coding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the post-transcriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in apoplastic wash fluids, GLYCINE-RICH RNA BINDING PROTEIN 7 (GRP7), as wells as the small RNA-binding protein ARGONAUTE2 (AGO2). These two proteins co-immunoprecipitated with each other, and with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of apoplastic wash fluid, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs.
Project description:Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. To downregulate immune responses and promote host colonization, U. maydis secretes a set of effector proteins into the plant apoplast. An effector essential for U. maydis virulence is Pit2, an inhibitor of papain-like cysteine proteases (PLCPs). Pit2 virulence function relies on a 14 amino acids motif (PID14). While sequence of the Pit2 effector is highly diverse amongst related pathogen species, the PID14 motif is highly conserved. Interestingly, synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. In line with this finding, mass spectrometry showed processing of Pit2 by maize PLCPs, which releases an inhibitory core motif of the PID14. Mutational analysis demonstrated that two residues of the released inhibitor peptide are essential for Pit2 function and, consequently, for U. maydis virulence. Based on these findings, we propose a model, in which the Pit2 effector functions as a decoy: Pit2 represents a favorable substrate for apoplastic PLCPs, which are central hubs of the maize immune system. Processing of Pit2 releases the inhibitor peptide, which in turn efficiently blocks PLCPs to modulate host immunity.