Project description:Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated seiv (suppressor of ectopic xylem vessel cell differentiation induced by VND7) mutants, which are suppressor mutants of VND7-inducible xylem vessel cell differentiation. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), uncharacterized F-BOX protein, PUB36, and UBIQUITIN-SPECIFIC PROTEASE1, respectively. We also found the decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitinated levels from 492 and 180 protein groups were up- and down-regulated, respectively. Proteins for cell wall biosynthesis and protein transport were ubiquitinated by the VND7 induction, whereas such active protein ubiquitination was not observed in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.
Project description:Direct target genes of VND7 were explored with inducible expression system using glucocorticoid receptor (GR). Transgenic plants expressing 35S:VND7-VP16-GR were treated with dexamethazone (DEX) and/or protein synthesis inhibitor cycloheximide (CHX). A number of genes related to the formation of vascular vessel was induced by DEX even in the presence of CHX. Total RNAs of the transgenic plants expressing 35S:VND7-VP16-GR treated with DEX plus CHX and those treated with CHX only were compared. As a control experiment, transgenic plants harboring empty vector were treated similarly and the total RNAs were compared similarly to identify genes merely induced by DEX treatment itself.
Project description:Plant immunity protects plants from numerous potentially pathogenic microbes. The biological network that controls plant inducible immunity must function effectively even when network components are targeted and disabled by pathogen effectors. Network buffering could confer this robustness by allowing different parts of the network to compensate for loss of each other’s functions. Networks rich in buffering rely on interactions within the network, but these mechanisms are difficult to study by simple genetic means. Through a network reconstitution strategy, where we disassemble and stepwise reassemble the plant immune network that mediates Pattern-Triggered-Immunity, we have resolved systems-level regulatory mechanisms underlying the Arabidopsis transcriptome response to the immune stimulant flagellin-22 (flg22). These mechanisms show widespread evidence of interactions among major sub-networks—the components that we call sectors—in the flg22-responsive transcriptome. Many of these interactions result in network buffering. Resolved regulatory mechanisms also show unexpected patterns for how the jasmonate (JA), ethylene (ET), phytoalexin-deficient 4 (PAD4), and salicylate (SA) signaling sectors control the transcriptional response to flg22. We demonstrate that many of these mechanisms are hidden from the traditional genetic approach of single-gene null-mutant analysis. As potential pathogenic perturbations to the network, null-mutant effects can be buffered by the immune network as well.
Project description:Lateral root organogenesis plays an essential role in defining plant root system architecture. In Arabidopsis, the AP2-family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify downstream targets of PUCHI, we engineered a transgenic line with inducible PUCHI activity by expressing a fusion protein of PUCHI and rat glucocorticoid receptor (GR) under the control of its own regulatory region (gPUCHI-GR) in the puchi-1 mutant.
Project description:Investigating putative RPS4-chromatin associations during Arabidopsis plant disease resistance, using a temperature-inducible system which resembles pathogen-infection
Project description:In this research a high-throughput RNA sequencing based transcriptome analysis technique (RNA-Seq) was used to evaluate differentially expressed genes (DEGs) in the wild type Arabidopsis seedling in response to flg22, a well-known microbe-associated molecular patterns (MAMP), and AtPep1, a well-known peptide representing an endogenous damage-associated molecular patterns (DAMP). The results of our study revealed that 1895 (1634 up-regulated and 261 down-regulated) and 2271 (1706 up-regulated and 565 down-regulated) significant differentially expressed genes in response to flg22 and AtPep1 treatment, respectively. Among significant DEGs, we observed that a number of hitherto overlooked genes have been found to be induced upon treatment with either flg22 or with AtPep1, indicating their possible involvement in innate immunity. Here, we characterized two of them, namely PP2-B13 and ACLP1. PP2-B13 contains an F-box domain and shows similarity to carbohydrate binding proteins. ACLP1 is a protein of unknown function with highest similarity to actin cross linking proteins and includes a fascin domain. Using qPCR, we verified that the genes encoding PP2-B13, and ACLP1 were highly induced upon treatment of leaf disks with flg22. We obtained T-DNA insertion mutants and generated homozygous mutant lines. None of the mutants showed a phenotype in the absence of infection. pp2-b13 and aclp1 mutants showed an increased susceptibility to infection by the virulent pathogen Pseudomomas syringae pv tomato mutant hrcC-, as evidenced by an increased growth of the pathogen in planta. Further we present evidence that aclp1 was deficient in ethylene production upon flg22 treatment, while pp2-b13, was deficient in ROS production. In conclusion, the products of these genes contribute to plant immunity against bacterial pathogens, although there is currently no clue for their mechanism of action. The results from this research provide new information to a better understanding of the immune system in Arabidopsis.
Project description:The goal of this experiment was to identify the early responsive genes activated by the 22 amino acid peptide of bacterial flagellin (flg22) in Arabidopsis seedlings that are involved in the initial responses important for plant innate immunity. We used the microarrays to detail the global program of gene expression underlying intial cellular and molecular responses to microbial associated molecualr patterns (MAMPs). Arabidopsis seedlings were treated with 2 nM flg22 for 30 min and 60 min. Transcript profiles were analyzed and compared between control and treated cells to identify early flg22 responsive genes.
Project description:Purpose: The goals of this study are to analyze the transcriptome of Arabidopsis thaliana treated after flg22 and to find changes of glucosinolate metabolism genes after treatment. Methods: Total mRNA of 10 day wild-type Arabidopsis seedlings that were treated with and without flg22( final concentrations 1μmol/L) in 1/2 MS medium for 4h, were extracted respectively. Each sample was harvested in three independent biological replicates with equal weight and mixed ,subsequently sequencing. The sequence reads that passed quality filters were mapped to the Arabidopsis_thaliana genome (TAIR10.18).Then the mapping genes were used for the abundance and functional analysis. Results: We mapped about 45 million and 52 million sequence reads of control sample and treatment sample to the Arabidopsis_thaliana genome (TAIR10.18) and identified total 23,413 genes with Botiw/TopHat workflow. Comparison of the two samples showed 1,200 differentially expressed genes (DEGs), including 290 down-regulated and 910 up-regulated genes. The DEGs were associated with energy metabolism, amino acid metabolism and biosynthesis of secondary metabolites. After flg22 treatment, genes involved in indolic glucosinolate biosynthesis pathway were up-regulated significantly,which is further demonstrated by Real Time RT-PCR, while aliphatic glucosinolate pathway almost had no change, indicating the important role of indolic glucosinolates in plant defense responses. Conclusion: Our study provides the overall genetic resource of Arabidopsis_thaliana after treated by flg22 to date. These data will pave the way for further studies about deeply understand pathogen induced defense and the contribution of indolic glucosinolates.
Project description:The goal of this experiment was to identify the early responsive genes activated by the 22 amino acid peptide of bacterial flagellin (flg22) in Arabidopsis mesophyll cells that are involved in the initial responses important for plant innate immunity. We used the microarrays to detail the global program of gene expression underlying intial cellular and molecular responses to microbial associated molecualr patterns (MAMPs). Arabidopsis mesophyll protoplasts were treated with 100 nM flg22 for 30 min and 60 min. Transcript profiles were analyzed and compared between control and treated cells to identify early flg22 responsive genes.