Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division

11 Pág.Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.This work was supported by the NSF Graduate Research Fellowship Program (DGE-1252376, to N.M.C. and A.P.F.). Research in the R. Simon lab was funded by the Deutsche Forschungsge-meinschaft (Si947/10 and an Alexander von Humboldt Foundation fellowship, to B.B.). This work was also supported by a grant from the Ministerio de Economía y Competitividad of Spain and European Regional Development Fund (BFU2016-80315-P, to M.A.M.-R.). E.B.A. is supported by Ayudante de Investigacion contract PEJ-2017-AI/BIO-7360 from Comunidad Madrid. S.G.Z. was supported by the HHMI and by a grant from the NIH (GM118036). Research in the K.L.G. lab was funded by NSF Grant 1243945. The R. Sozzani lab is supported by an NSF CAREER grant (MCB-1453130) and the North Carolina Agricultural & Life Sciences Research Foundation at North Carolina State University’s College of Agricultural and Life Sciences.Peer reviewe

Nuclear Pore Permeabilization Is a Convergent Signaling Event in Effector-Triggered Immunity

Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction

A Noncanonical Role for the CKI-RB-E2F Cell-Cycle Signaling Pathway in Plant Effector-Triggered Immunity

Effector-triggered immunity (ETI), the major host defense mechanism in plants, is often associated with programmed cell death (PCD). Plants lack close homologs of caspases, the key mediators of PCD in animals. So although the NB-LRR receptors involved in ETI are well studied, how they activate PCD and confer disease resistance remains elusive. We show that the Arabidopsis nuclear envelope protein, CPR5, negatively regulates ETI and the associated PCD through a physical interaction with cyclin-dependent kinase inhibitors (CKIs). Upon ETI induction, CKIs are released from CPR5 to cause overactivation of another core cell-cycle regulator, E2F. In cki and e2f mutants, ETI responses induced by both TIR-NB-LRR and CC-NB-LRR classes of immune receptors are compromised. We further show that E2F is deregulated during ETI, probably through CKI-mediated hyperphosphorylation of retinoblastoma-related 1 (RBR1). This study demonstrates that canonical cell-cycle regulators also play important noncanonical roles in plant immunity

A transcriptomics approach uncovers novel roles for poly(ADP-ribosyl)ation in the basal defense response in Arabidopsis thaliana.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) or loss of Arabidopsis thaliana PARG1 (poly(ADP-ribose) glycohydrolase) disrupt a subset of plant defenses. In the present study we examined the impact of altered poly(ADP-ribosyl)ation on early gene expression induced by the microbe-associate molecular patterns (MAMPs) flagellin (flg22) and EF-Tu (elf18). Stringent statistical analyses and filtering identified 178 genes having MAMP-induced mRNA abundance patterns that were altered by either PARP inhibitor 3-aminobenzamide (3AB) or PARG1 knockout. From the identified set of 178 genes, over fifty Arabidopsis T-DNA insertion lines were chosen and screened for altered basal defense responses. Subtle alterations in callose deposition and/or seedling growth in response to those MAMPs were observed in knockouts of At3g55630 (FPGS3, a cytosolic folylpolyglutamate synthetase), At5g15660 (containing an F-box domain), At1g47370 (a TIR-X (Toll-Interleukin Receptor domain)), and At5g64060 (a predicted pectin methylesterase inhibitor). Over-represented GO terms for the gene expression study included "innate immune response" for elf18/parg1, highlighting a subset of elf18-activated defense-associated genes whose expression is altered in parg1 plants. The study also allowed a tightly controlled comparison of early mRNA abundance responses to flg22 and elf18 in wild-type Arabidopsis, which revealed many differences. The PARP inhibitor 3-methoxybenzamide (3MB) was also used in the gene expression profiling, but pleiotropic impacts of this inhibitor were observed. This transcriptomics study revealed targets for further dissection of MAMP-induced plant immune responses, impacts of PARP inhibitors, and the molecular mechanisms by which poly(ADP-ribosyl)ation regulates plant responses to MAMPs

Experimental design.

<p><b>A.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) seedlings were pre-treated for two hours with either 3AB, 3-MB, or vehicle (DMSO), and then treated for one hour with either flg22 flagellin peptide or sterile H₂O. <b>B.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) or <i>parg1-2</i> knockout seedlings were treated for one hour with either elf18 EF-TU peptide or sterile H₂O.</p

Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.

<p>Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.</p

Seedling growth inhibition assay.

<p>Five-day-old seedlings of the indicated genotypes were treated with 0.05uM (low) or 1.0uM (high) flg22 peptide and grown for an additional 14 d. Fresh seedlings weights were then recorded and normalized to the average untreated weight within each genotype. A. Pectin methylesterase inhibitor (<i>PMEI</i>) (At5g64640) knockouts versus untreated, three (<i>pmei-1</i>) and four (<i>pmei-2</i>) biological replicates of 12 seedlings per treatment. B. F-box domain-containing gene (At5g15660) knockouts versus untreated, three (<i>f-box-1</i>) and two (<i>f-box-2</i>) biological replicates of 12 seedlings each. C. <i>Folylpolygutamate synthetase 3 (FPGS3)</i> (At3g55630) knockout versus untreated, three biological replicates. D. TIR-X domain-containing gene (At1g47370), three biological replicates. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with the same concentration of flg22. E. <i>FPGS1</i> (At5g05980), <i>FPGS2</i> (At3g10160), and <i>FPGS3</i> (At3g55630) versus untreated, three biological replicates. (Tukey's simultaneous test: * <i>P</i> < 0.05; **<i>P</i> < 0.005; no asterisk, <i>P</i> > 0.05).</p

Determination of differentially regulated genes.

<p>After initial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190268#sec002" target="_blank">methods</a>), lists of genes differentially regulated between flg22 and flg22 + 3AB (<b>A-D</b>) or between Col-0 elf18 and <i>parg1-2</i> elf18 (<b>E-H</b>) were assembled. Upregulated (red) (<b>A, C, E, F</b>) and downregulated (blue) (<b>B, D, G, H</b>) genes were determined separately. “Broken” genes were defined for this study as those genes that displayed statistically significant differences in mRNA abundance after treatment with MAMP (flg22 or elf18) (stringent cutoff = FDR <0.05, fold-change>1.3 or <-1.3) versus untreated, but for which MAMP treatment in the presence of 3AB or <i>parg1-2</i> did not cause a statistically significant difference (even at the relatively permissive cutoff of p<0.05, fold-change>1.0 or <-1.0). “Misregulated” genes were defined as those genes upon which MAMPs had no statistically significant impact on mRNA abundance in wild-type plants, even using non-stringent cutoff values (p<0.05 and fold-change>1.0 or <-1.0), but for which MAMP treatment did cause significant differences (at stringent cutoff values) in the presence of either 3AB or <i>parg1-2</i>. To further reduce the occurrence of Type I false positives, the highlighted gene lists were then filtered once more for flg22 v flg22 + 3AB (p<0.05, fold-change>1.3 or <-1.3) or Col-0 elf18 v <i>parg1-2</i> elf18 (p<0.05, fold-change>1.3 or <-1.3).</p

Hierarchical clustering of expression patterns for all 30,387 genes present on 1-plex Nimblegen <i>Arabidopsis thaliana</i> array.

<p>Standardized transcript abundances (mean = 0, standard deviation = 1) for three biological replicates of nine treatments (Col-0 untreated, Col-0 + flg22, Col-0 + 3AB, Col-0 + 3MB, Col-0 + flg22 + 3AB, Col-0 + flg22 + 3MB, Col-0 + elf18, <i>parg1-2</i> untreated, and <i>parg1-2</i> + elf18) were used to determine Euclidean distances between treatments and genes, represented by the left and top dendrograms, respectively. f = 1μM flg22, e = 1μM elf18, A = 2.5mM 3-aminobenzmide (3AB), M = 2.5mM 3-methoxybenzamide (3MB), C = Col-0 (wild-type), p = <i>parg1-2</i>.</p

Callose deposition assay.

<p>A. 10-day-old Arabidopsis seedlings were treated with distilled, deionized water (H₂O) or 1 μm flg22, fixed 24 h after flg22 elicitation, and visualized for callose deposition by aniline blue staining and epifluorescence microscopy. Degree of callose deposition was categorized using a scale of 0 to 5, 0 = no callose deposits, 5 = dense callose deposits over entire field of view. Twelve cotyledons per genotype were examined and compared to wild-type (Col-0) responses per biological replicate (n). <i>pmei-2</i> (At5g64640) n = 1; <i>rba-2</i> (At1g47370) n = 5; <i>f-box-1</i> (At5g15660) n = 4; <i>fpgs3-1</i> and <i>fpgs3-2</i> (At3g55630) n = 4, n = 1, respectively. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with flg22 (Tukey's simultaneous test: †P<0.15; ††P<0.1; *P<0.05; **P<0.005; no asterisk, P > 0.05). B. For selected lines, callose deposits were quantified as average percent area covered by white pixels within the viewfield, corresponding to flg22-induced callose, +/- standard error. Ten cotyledons per genotype were examined for each of four biological replicates. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with flg22 (Tukey's simultaneous test: *P < 0.055; **P < 0.005; no asterisk, P > 0.05). Representative flg22-treated cotyledons for each genotype in B are shown in C.</p