In JoVE (1)

Other Publications (15)

Articles by Shauna C. Somerville in JoVE

Other articles by Shauna C. Somerville on PubMed

PMR6, a Pectate Lyase-like Gene Required for Powdery Mildew Susceptibility in Arabidopsis

The Plant Cell. Sep, 2002  |  Pubmed ID: 12215508

The plant genes required for the growth and reproduction of plant pathogens are largely unknown. In an effort to identify these genes, we isolated Arabidopsis mutants that do not support the normal growth of the powdery mildew pathogen Erysiphe cichoracearum. Here, we report on the cloning and characterization of one of these genes, PMR6. PMR6 encodes a pectate lyase-like protein with a novel C-terminal domain. Consistent with its predicted gene function, mutations in PMR6 alter the composition of the plant cell wall, as shown by Fourier transform infrared spectroscopy. pmr6-mediated resistance requires neither salicylic acid nor the ability to perceive jasmonic acid or ethylene, indicating that the resistance mechanism does not require the activation of well-described defense pathways. Thus, pmr6 resistance represents a novel form of disease resistance based on the loss of a gene required during a compatible interaction rather than the activation of known host defense pathways.

MLO, a Novel Modulator of Plant Defenses and Cell Death, Binds Calmodulin

Trends in Plant Science. Sep, 2002  |  Pubmed ID: 12234724

Expression Profile Analysis of the Low-oxygen Response in Arabidopsis Root Cultures

The Plant Cell. Oct, 2002  |  Pubmed ID: 12368499

We used DNA microarray technology to identify genes involved in the low-oxygen response of Arabidopsis root cultures. A microarray containing 3500 cDNA clones was screened with cDNA samples taken at various times (0.5, 2, 4, and 20 h) after transfer to low-oxygen conditions. A package of statistical tools identified 210 differentially expressed genes over the four time points. Principal component analysis showed the 0.5-h response to contain a substantially different set of genes from those regulated differentially at the other three time points. The differentially expressed genes included the known anaerobic proteins as well as transcription factors, signal transduction components, and genes that encode enzymes of pathways not known previously to be involved in low-oxygen metabolism. We found that the regulatory regions of genes with a similar expression profile contained similar sequence motifs, suggesting the coordinated transcriptional control of groups of genes by common sets of regulatory factors.

Systemic Gene Expression in Arabidopsis During an Incompatible Interaction with Alternaria Brassicicola

Plant Physiology. Jun, 2003  |  Pubmed ID: 12805628

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of "high alert," otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the beta-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Loss of a Callose Synthase Results in Salicylic Acid-dependent Disease Resistance

Science (New York, N.Y.). Aug, 2003  |  Pubmed ID: 12920300

Plants attacked by pathogens rapidly deposit callose, a beta-1,3-glucan, at wound sites. Traditionally, this deposition is thought to reinforce the cell wall and is regarded as a defense response. Surprisingly, here we found that powdery mildew resistant 4 (pmr4), a mutant lacking pathogen-induced callose, became resistant to pathogens, rather than more susceptible. This resistance was due to mutation of a callose synthase, resulting in a loss of the induced callose response. Double-mutant analysis indicated that blocking the salicylic acid (SA) defense signaling pathway was sufficient to restore susceptibility to pmr4 mutants. Thus, callose or callose synthase negatively regulates the SA pathway.

SNARE-protein-mediated Disease Resistance at the Plant Cell Wall

Nature. Oct, 2003  |  Pubmed ID: 14586469

Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.

Mutations in PMR5 Result in Powdery Mildew Resistance and Altered Cell Wall Composition

The Plant Journal : for Cell and Molecular Biology. Dec, 2004  |  Pubmed ID: 15584961

Powdery mildews and other obligate biotrophic pathogens are highly adapted to their hosts and often show limited host ranges. One facet of such host specialization is likely to be penetration of the host cell wall, a major barrier to infection. A mutation in the pmr5 gene rendered Arabidopsis resistant to the powdery mildew species Erysiphe cichoracearum and Erysiphe orontii, but not to the unrelated pathogens Pseudomonas syringae or Peronospora parasitica. PMR5 belongs to a large family of plant-specific genes of unknown function. pmr5-mediated resistance did not require signaling through either the salicylic acid or jasmonic acid/ethylene defense pathways, suggesting resistance in this mutant may be due either to the loss of a susceptibility factor or to the activation of a novel form of defense. Based on Fourier transform infrared analysis, the pmr5 cell walls were enriched in pectin and exhibited a reduced degree of pectin modification relative to wild-type cell walls. In addition, the mutant had smaller cells, suggesting a defect in cell expansion. A double mutant with pmr6 (defective in a glycosylphosphatidylinositol-anchored pectate lyase-like gene) exhibited a strong increase in total uronic acid content and a more severe reduction in size, relative to the single mutants, suggesting that the two genes affect pectin composition, either directly or indirectly, via different mechanisms. These two mutants highlight the importance of the host cell wall in plant-microbe interactions.

Conserved Requirement for a Plant Host Cell Protein in Powdery Mildew Pathogenesis

Nature Genetics. Jun, 2006  |  Pubmed ID: 16732289

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.

Focal Accumulation of Defences at Sites of Fungal Pathogen Attack

Journal of Experimental Botany. 2008  |  Pubmed ID: 18703493

Plants resist attack by haustorium-forming biotrophic and hemi-biotrophic fungi through fortification of the cell wall to prevent penetration through the wall and the subsequent establishment of haustorial feeding structures by the fungus. While the existence of cell wall-based defences has been known for many years, only recently have the molecular components contributing to such defences been identified. Forward genetic screens identified Arabidopsis mutants impaired in penetration resistance to powdery mildew fungi that were normally halted at the cell wall. Several loci contributing to penetration resistance have been identified and a common feature is the striking focal accumulation of proteins associated with penetration resistance at sites of interaction with fungal appressoria and penetration pegs. The focal accumulation of defence-related proteins and the deposition of cell wall reinforcements at sites of attempted fungal penetration represent an example of cell polarization and raise many questions of relevance, not only to plant pathology but also to general cell biology.

A Lesion-mimic Syntaxin Double Mutant in Arabidopsis Reveals Novel Complexity of Pathogen Defense Signaling

Molecular Plant. May, 2008  |  Pubmed ID: 19825557

The lesion-mimic Arabidopsis mutant, syp121 syp122, constitutively expresses the salicylic acid (SA) signaling pathway and has low penetration resistance to powdery mildew fungi. Genetic analyses of the lesion-mimic phenotype have expanded our understanding of programmed cell death (PCD) in plants. Inactivation of SA signaling genes in syp121 syp122 only partially rescues the lesion-mimic phenotype, indicating that additional defenses contribute to the PCD. Whole genome transcriptome analysis confirmed that SA-induced transcripts, as well as numerous other known pathogen-response transcripts, are up-regulated after inactivation of the syntaxin genes. A suppressor mutant analysis of syp121 syp122 revealed that FMO1, ALD1, and PAD4 are important for lesion development. Mutant alleles of EDS1, NDR1, RAR1, and SGT1b also partially rescued the lesion-mimic phenotype, suggesting that mutating syntaxin genes stimulates TIR-NB-LRR and CC-NB-LRR-type resistances. The syntaxin double knockout potentiated a powdery mildew-induced HR-like response. This required functional PAD4 but not functional SA signaling. However, SA signaling potentiated the PAD4-dependent HR-like response. Analyses of quadruple mutants suggest that EDS5 and SID2 confer separate SA-independent signaling functions, and that FMO1 and ALD1 mediate SA-independent signals that are NPR1-dependent. These studies highlight the contribution of multiple pathways to defense and point to the complexity of their interactions.

Sugar Transporters for Intercellular Exchange and Nutrition of Pathogens

Nature. Nov, 2010  |  Pubmed ID: 21107422

Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Despite broad biological importance, the identity of sugar efflux transporters has remained elusive. Using optical glucose sensors, we identified a new class of sugar transporters, named SWEETs, and show that at least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport. Arabidopsis SWEET8 is essential for pollen viability, and the rice homologues SWEET11 and SWEET14 are specifically exploited by bacterial pathogens for virulence by means of direct binding of a bacterial effector to the SWEET promoter. Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is probably targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells.

Visualizing Cellular Dynamics in Plant-microbe Interactions Using Fluorescent-tagged Proteins

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21359815

Interactions between plant cells and microbial pathogens involve highly dynamic processes of cellular trafficking and reorganization. Substantial advances in imaging technologies, including the discovery and widespread use of fluorescent proteins as tags as well as advances in laser-based confocal microscopy have provided the first glimpses of the dynamic nature of the processes of defense and pathogenicity. Prior to the development of these techniques, high resolution imaging by electron microscopy gave only a static picture of these dynamic events and live cell imaging was significantly limited in resolution as well as the availability of relevant stains and markers. The incorporation of fluorescent protein fusions and laser-based confocal microscopy into studies of plant-microbe interactions has opened the door to fascinating new questions about the cellular response to attempted infection. Additionally, studies of cellular responses to pathogen infection may lead to new knowledge about fundamental processes in plant cells, such as mechanisms underlying subcellular trafficking and targeting of proteins and other molecules.

Elevated Early Callose Deposition Results in Complete Penetration Resistance to Powdery Mildew in Arabidopsis

Plant Physiology. Mar, 2013  |  Pubmed ID: 23335625

A common response by plants to fungal attack is deposition of callose, a (1,3)-β-glucan polymer, in the form of cell wall thickenings called papillae, at site of wall penetration. While it has been generally believed that the papillae provide a structural barrier to slow fungal penetration, this idea has been challenged in recent studies of Arabidopsis (Arabidopsis thaliana), where fungal resistance was found to be independent of callose deposition. To the contrary, we show that callose can strongly support penetration resistance when deposited in elevated amounts at early time points of infection. We generated transgenic Arabidopsis lines that express POWDERY MILDEW RESISTANT4 (PMR4), which encodes a stress-induced callose synthase, under the control of the constitutive 35S promoter. In these lines, we detected callose synthase activity that was four times higher than that in wild-type plants 6 h post inoculation with the virulent powdery mildew Golovinomyces cichoracearum. The callose synthase activity was correlated with enlarged callose deposits and the focal accumulation of green fluorescent protein-tagged PMR4 at sites of attempted fungal penetration. We observed similar results from infection studies with the nonadapted powdery mildew Blumeria graminis f. sp. hordei. Haustoria formation was prevented in resistant transgenic lines during both types of powdery mildew infection, and neither the salicylic acid-dependent nor jasmonate-dependent pathways were induced. We present a schematic model that highlights the differences in callose deposition between the resistant transgenic lines and the susceptible wild-type plants during compatible and incompatible interactions between Arabidopsis and powdery mildew.

Perception of Conserved Pathogen Elicitors at the Plasma Membrane Leads to Relocalization of the Arabidopsis PEN3 Transporter

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2013  |  Pubmed ID: 23836668

The Arabidopsis penetration resistance 3 (PEN3) ATP binding cassette transporter participates in nonhost resistance to fungal and oomycete pathogens and is required for full penetration resistance to the barley powdery mildew Blumeria graminis f. sp. hordei. PEN3 resides in the plasma membrane and is recruited to sites of attempted penetration by invading fungal appressoria, where the transporter shows strong focal accumulation. We report that recruitment of PEN3 to sites of pathogen detection is triggered by perception of pathogen-associated molecular patterns, such as flagellin and chitin. PEN3 recruitment requires the corresponding pattern recognition receptors but does not require the BAK1 coreceptor. Pathogen- and pathogen-associated molecular pattern-induced focal accumulation of PEN3 and the penetration resistance 1 (PEN1) syntaxin show differential sensitivity to specific pharmacological inhibitors, indicating distinct mechanisms for recruitment of these defense-associated proteins to the host-pathogen interface. Focal accumulation of PEN3 requires actin but is not affected by inhibitors of microtubule polymerization, secretory trafficking, or protein synthesis, and plasmolysis experiments indicate that accumulation of PEN3 occurs outside of the plasma membrane within papillae. Our results implicate pattern recognition receptors in the recruitment of defense-related proteins to sites of pathogen detection. Additionally, the process through which PEN3 is recruited to the host-pathogen interface is independent of new protein synthesis and BFA-sensitive secretory trafficking events, suggesting that existing PEN3 is redirected through an unknown trafficking pathway to sites of pathogen detection for export into papillae.

Interaction of the Arabidopsis GTPase RabA4c with Its Effector PMR4 Results in Complete Penetration Resistance to Powdery Mildew

The Plant Cell. Jul, 2014  |  Pubmed ID: 25056861

The (1,3)-β-glucan callose is a major component of cell wall thickenings in response to pathogen attack in plants. GTPases have been suggested to regulate pathogen-induced callose biosynthesis. To elucidate the regulation of callose biosynthesis in Arabidopsis thaliana, we screened microarray data and identified transcriptional upregulation of the GTPase RabA4c after biotic stress. We studied the function of RabA4c in its native and dominant negative (dn) isoform in RabA4c overexpression lines. RabA4c overexpression caused complete penetration resistance to the virulent powdery mildew Golovinomyces cichoracearum due to enhanced callose deposition at early time points of infection, which prevented fungal ingress into epidermal cells. By contrast, RabA4c(dn) overexpression did not increase callose deposition or penetration resistance. A cross of the resistant line with the pmr4 disruption mutant lacking the stress-induced callose synthase PMR4 revealed that enhanced callose deposition and penetration resistance were PMR4-dependent. In live-cell imaging, tagged RabA4c was shown to localize at the plasma membrane prior to infection, which was broken in the pmr4 disruption mutant background, with callose deposits at the site of attempted fungal penetration. Together with our interactions studies including yeast two-hybrid, pull-down, and in planta fluorescence resonance energy transfer assays, we concluded that RabA4c directly interacts with PMR4, which can be seen as an effector of this GTPase.

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