Project description:To decipher gene expression controlled by the five highly homologous group S1 bZIP transcription factors during the reproductive growth phase of Arabidopsis thaliana, we generated triple (bzip2/-11/-44) and quintuple (bzip1/-2/-11/-44/-53) mutants of these factors using CRISPR/Cas9 and analysed gene expression in distinct C source (source leaves) or C sink (sink leaves, rosette buds, flowers) tissues.
Project description:In compatible interactions, biotrophic microbial phytopathogens rely on the supply of carbon and nitrogen assimilates by the colonized host tissue. Successful biotrophs need to reprogram host metabolism, which also involves the stimulation of assimilate export from living host cells into the plant-pathogen interface at the infection site. In rice and cassava, SWEET sucrose transporters, are induced by bacterial TAL (transcriptional activator-like) effectors to establish compatibility. A pathogen-specific transcriptional induction of SWEET transporters has also been observed in Arabidopsis leaves upon microbial challenge. Here, we have assessed the question, whether the phloem localized AtSWEET11 and AtSWEET12 transporters represent susceptibility factors in the interaction of Arabidopsis with the fungal hemibiotroph Colletotrichum higginsianum (Ch). Compared to wild type, sweet11/sweet12 double mutants exhibited priming of the SA pathway in mock conditions. To investigate transcriptional changes in C. higgsinanum infected leaves, five-week old Arabidopsis plants were spray infected with 2 Mio. conidia/ ml 1h before lights off and fully expanded leaves of wild type Col-0 and the sweet11/sweet12 double mutant were harvested in three situations: 1) immediately before treatment, 2) from mock treated plants (sprayed with water) at 2.5 days post treatment and 3) from C. higginsianum inoculated leaves during biotrophic colonization at 2.5 days post treatment
Project description:In compatible interactions, biotrophic microbial phytopathogens rely on the supply of carbon and nitrogen assimilates by the colonized host tissue. Successful biotrophs need to reprogram host metabolism, which also involves the stimulation of assimilate export from living host cells into the plant-pathogen interface at the infection site. In rice and cassava, SWEET sucrose transporters, are induced by bacterial TAL (transcriptional activator-like) effectors to establish compatibility. A pathogen-specific transcriptional induction of SWEET transporters has also been observed in Arabidopsis leaves upon microbial challenge. Here, we have assessed the question, whether the phloem localized AtSWEET11 and AtSWEET12 transporters represent susceptibility factors in the interaction of Arabidopsis with the fungal hemibiotroph Colletotrichum higginsianum (Ch). Compared to wild type, sweet11/sweet12 double mutants exhibited priming of the SA pathway in mock conditions.
Project description:Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Post-translational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B’γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against UV-B or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.
Project description:Seedlings of Arabidopsis wild type and ATE1/2 double knock-out mutants were treated with flagellin 22 peptide to elicit pathogen-triggered immunity (PTI) responses. Leaves were harvested and proteome composition assessed.
Project description:Arabidopsis thaliana transcriptome analysis in response to plant growth promoting rhizobacteria (PGPR)<br> Experiment 1 : Changes in gene expression profile triggered during root architecture response to Phyllobacterium.<br> Biological question : Which genes are up- or down-regulated in Arabidopsis thaliana cultivated in vitro with increased lateral root development in response to Phyllobacterium STM196 inoculation.<br> Experiment description: Seeds of wild-type Arabidopsis thaliana (ecotype Columbia) were surface-sterilized and sown on agar mineral medium (see below). 4 days after storage in the dark at 4C, seedling were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on a fresh agar mineral medium inoculated or not with Phyllobacterium STM196 (2.108 cfu/ml). 6 days later, root and leaves were collected, froze on liquid nitrogen and stored at -80C.<br> <br> Experiment 2 : Changes in gene expression profile triggered during induced systemic resistance (ISR)<br> Biological question : Which genes are up- or down-regulated during the ISR triggered by a rhizobacteria, in comparison with those affected by a pathogenic interaction. <br> Experiment description: Seeds were sown on 0.8% (W/V) agar mineral medium (see below). 4 days after storage in the dark at 4C, seedling were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on soil inoculated or not with 107 cfu.g-1 of Bradyrhizobium strain ORS278. Three weeks later, 3 leaves per plant were infiltrated with a suspension of Pseudomonas syringae pv. tomato (2.105 cfu.ml-1) or with MgSO4 10 mM alone for control plants. Infiltrated leaves were collected 24h later.<br> <br> Experiment 3 : Comparison of the effects of 3 rhizobacteria on Arabidopsis thaliana transcriptome<br> Biological question : which genes are specifically induced or repressed in Arabidopsis thaliana by inoculation of the soil with a PGPR vs a bacteria that has the ability to trigger nodule formation in a Legume. <br> Experiment description: Seeds of wild-type Arabidopsis thaliana (ecotype Columbia) were surface-sterilized and sown on agar mineral medium. Four days after storage in the dark at 4C, seedlings were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on soil inoculated or not with 108 cfu.g-1 of Mesorhizobium loti, or 108 cfu.g-1 of Phyllobacterium STM196, or 107 cfu.g-1 of Bradyrhizobium ORS278.
Project description:The characteristic leaf shapes we see in all plants are in good part outcome of the combined action of several transcription factor networks that translate into cell division activity during the early development of the organ. We show here that wild-type leaves have distinct transcriptomic profiles in center and marginal regions. Certain transcripts are enriched in margins, including those of CINCINNATA-like TCPs, and members of the NGATHA (NGA) and STYLISH (STY) gene families. We study in detail the contribution of miR319 regulated TCP (Teosinte branched, Cycloidea, PCF1/2) transcription factors to the development of the center and marginal regions of Arabidopsis leaves. We compare in molecular analyses wildtype, a tcp2 tcp4 mutant that has enlarged flat leaves and a tcp2 tcp3 tcp4 tcp10 mutant with strongly crinkled leaves. The different leaf domains of the tcp mutants show changed expression patterns for many photosynthesis related genes, indicating delayed differentiation, especially in the marginal parts of the organ. At the same time, we found an upregulation of cyclin genes and other genes that are known to participate in cell division, specifically in the marginal regions of tcp2 tcp3 tcp4 tcp10. Using GUS reporter constructs we confirmed extended mitotic activity in the tcp2 tcp3 tcp4 tcp10 leaf which persisted in small defined foci in the margins when the mitotic activity had already ceased in wild-type leaves. Our results describe the role of miR319-regulated TCP transcription factors in the coordination of activities in different leaf domains during the organs development.
Project description:Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists, mainly for technical reasons, on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGome generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, most OGs were acetyl- and methylesterified, and 80 % of them were produced by fungal pectin lyases (PNL), not polygalacturonases (PG). PG products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGomes and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence respectively, identified novel candidate OG elicitors. In conclusion, OGome analysis provides new insights into the enzymatic arms race between plant and pathogen and facilitates the identification of novel defense elicitors.
Project description:The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 hours post inoculation. Proteins were TCA-acetone-phenol extracted and subjected to 2-DE (5-8 pH range) and MS/MS (MALDI-TOF-TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana-Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 hours post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 hours post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 hours post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.