Project description:<p>Botrytis cinerea is a necrotrophic pathogen that induces host cell death, causing substantial economic losses in agriculture. In this study, we show that functional depletion of INOSITOL AUXOTROPHY 80 (INO80), a nuclear factor involved in local chromatin remodeling, reduces the susceptibility of model plant Arabidopsis to this necrotrophic pathogen. Using spatial transcriptomics, a cutting-edge technology enabling simultaneous acquisition of transcriptomes from distinct regions within the same tissue, we provide an unprecedented spatial profiling of the transcriptional response in Arabidopsis leaves following B. cinerea infection. Sequential changes in numerous key genes associated with plant defense pathways were observed across different regions of infected leaves. Additionally, by comparing the phosphoproteome and metabolome of wild-type (WT) and ino80 mutant leaves after B. cinerea infection, we reveal the role of INO80 in regulating multiple hormone pathways and metabolic processes, underscoring the critical importance of chromatin machinery in modulating plant responses to biotic stresses. Collectively, our findings establish a framework for describing, exploring and elucidating spatial information alongside plant multi-molecular responses upon encountering pathogenic challenges.</p>
Project description:The control of amount and activity of nuclear proteins is fundamental in regulating gene expression but is unstudied comprehensively in the adaptive response to pathogens. Highly specific labeling, enrichment and measurement of the nuclear proteome using TurboID LC-MS of Arabidopsis thaliana leaves treated with flg22 and/or cycloheximide was done. This discriminated translation followed by protein import, trafficking of preexisting protein, alleviation of repression and protein turn-over in the nucleus upon elicitation of basal immunity. Around 2,000 nuclear proteins were repeatedly quantified including more than 300 transcription factors or proteins related to transcription. Several proteins with documented activity in the endosome and MVBs were synthesized and imported upon PAMP challenge, suggesting alternative nuclear functions in PTI, mainly of an obscure cysteine/histidine rich protein family. Circadian clock components, particularly the transcription factor CHE were depleted upon PAMP challenge, suggesting a safeguard against untimely induction of SAR. Moonlighting proteins exercising alternative functions in the nucleus as well as protein trafficking and turn-over regulation are common in modulating the multi-facetted layers of plant immunity. The highly specific, deep coverage of proteins in the nucleus makes the study a resource for anyone interested in plant nuclear proteome dynamics and defense.
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: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:Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We exposed Arabidopsis to Pseudomonas syringae or mock treatment and profiled the transcriptomes of 11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from Pseudomonas and mock inoculated leaves identified five distinct pathogen responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection revealed a continuum of disease progression from an immune to susceptible state. Confocal imaging of promoter reporter lines for pathogen responsive cell clusters visualized immune activated cell clusters surrounding substomatal cavities colonized by bacteria, suggesting immune clusters are sites of early pathogen invasion. Susceptibility cell clusters exhibited more general localization and were highly induced at later stages of infection. Overall, our work uncovers cellular heterogeneity within an infected leaf and provides unique insight into plant differential response to infection at a single-cell level.
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.
Project description:Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We exposed Arabidopsis to Pseudomonas syringae or mock treatment and profiled the transcriptomes of 11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from Pseudomonas and mock inoculated leaves identified five distinct pathogen responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection revealed a continuum of disease progression from an immune to susceptible state. Confocal imaging of promoter reporter lines for pathogen responsive cell clusters visualized immune activated cell clusters surrounding substomatal cavities colonized by bacteria, suggesting immune clusters are sites of early pathogen invasion. Susceptibility cell clusters exhibited more general localization and were highly induced at later stages of infection. Overall, our work uncovers cellular heterogeneity within an infected leaf and provides unique insight into plant differential response to infection at a single-cell level.
Project description:Pathogen invasion in plants is associated with transcriptional reprogramming. Enigmatically, plants induce similar transcriptome responses upon infection by virulent or avirulent pathogens. This renders the importance of transcriptional reprogramming for immunity obscure. Here, using RNA-seq, we generate time-series transcriptome data coupled with genetic perturbations to reveal temporal dynamics upon infection by virulent or avirulent strains of a bacterial pathogen, Pseudomonas syringae, in Arabidopsis thaliana. Fast and sustained transcriptional reprogramming occurs upon infection with avirulent strains while virulent strain infection leads to a slower response with comparable gene expression patterns and magnitudes. Importantly, transcriptome analysis of resistant and susceptible mutants responding to avirulent strains links delayed transcriptional reprogramming to compromised immunity. Taken together, our results pinpoint the early critical time window of transcriptional reprogramming for establishing effective immunity against the bacterial pathogen.
Project description:We examined changes in steady-state transcript level in leaves of Arabidopsis plants subjected to salinity, heat stress and their combination by a transcriptome analysis of leaves.