Novel Disease Susceptibility Factors for Fungal Necrotrophic Pathogens in Arabidopsis

Host cells use an intricate signaling system to respond to invasions by pathogenic microorganisms. Although several signaling components of disease resistance against necrotrophic fungal pathogens have been identified, our understanding for how molecular components and host processes contribute to plant disease susceptibility is rather sparse. Here, we identified four transcription factors (TFs) from Arabidopsis that limit pathogen spread. Arabidopsis mutants defective in any of these TFs displayed increased disease susceptibility to Botrytis cinerea and Plectosphaerella cucumerina, and a general activation of non-immune host processes that contribute to plant disease susceptibility. Transcriptome analyses revealed that the mutants share a common transcriptional signature of 77 up-regulated genes. We characterized several of the up-regulated genes that encode peptides with a secretion signal, which we named PROVIR (for provirulence) factors. Forward and reverse genetic analyses revealed that many of the PROVIRs are important for disease susceptibility of the host to fungal necrotrophs. The TFs and PROVIRs identified in our work thus represent novel genetic determinants for plant disease susceptibility to necrotrophic fungal pathogens.Funding: This work was supported by the Spanish MINECO (BFU2012 to PV), and Generalitat Valenciana (Prometeo2014/020 to PV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Dobón Alonso, A.; Canet Perez, JV.; García-Andrade Serrano, J.; Angulo, C.; Neumetzler, L.; Persson, S.; Vera Vera, P. (2015). Novel Disease Susceptibility Factors for Fungal Necrotrophic Pathogens in Arabidopsis. PLoS Pathogens. 11(4):1-30. https://doi.org/10.1371/journal.ppat.1004800S13011

AXY8 Encodes an α-Fucosidase, Underscoring the Importance of Apoplastic Metabolism on the Fine Structure of Arabidopsis Cell Wall Polysaccharides[W][OA]

Characterization of an Arabidopsis thaliana mutant with an altered structure of the cell wall hemicellulose xyloglucan lead to the identification of an apoplastic fucosidase, AXY8. The data suggest that apoplastic trimming of cell wall polysaccharides by plant glycosylhydrolases plays a major role in generating the structural heterogeneity and complexity found in wall polysaccharides

TRICHOME BIREFRINGENCE and Its Homolog AT5G01360 Encode Plant-Specific DUF231 Proteins Required for Cellulose Biosynthesis in Arabidopsis1[W][OA]

The Arabidopsis (Arabidopsis thaliana) trichome birefringence (tbr) mutant has severely reduced crystalline cellulose in trichomes, but the molecular nature of TBR was unknown. We determined TBR to belong to the plant-specific DUF231 domain gene family comprising 46 members of unknown function in Arabidopsis. The genes harbor another plant-specific domain, called the TBL domain, which contains a conserved GDSL motif known from some esterases/lipases. TBR and TBR-like3 (TBL3) are transcriptionally coordinated with primary and secondary CELLULOSE SYNTHASE (CESA) genes, respectively. The tbr and tbl3 mutants hold lower levels of crystalline cellulose and have altered pectin composition in trichomes and stems, respectively, tissues generally thought to contain mainly secondary wall crystalline cellulose. In contrast, primary wall cellulose levels remain unchanged in both mutants as measured in etiolated tbr and tbl3 hypocotyls, while the amount of esterified pectins is reduced and pectin methylesterase activity is increased in this tissue. Furthermore, etiolated tbr hypocotyls have reduced length with swollen epidermal cells, a phenotype characteristic for primary cesa mutants or the wild type treated with cellulose synthesis inhibitors. Taken together, we show that two TBL genes contribute to the synthesis and deposition of secondary wall cellulose, presumably by influencing the esterification state of pectic polymers

Interactions between MUR10/CesA7-Dependent Secondary Cellulose Biosynthesis and Primary Cell Wall Structure

Primary cell walls are deposited and remodeled during cell division and expansion. Secondary cell walls are deposited in specialized cells after the expansion phase. It is presently unknown whether and how these processes are interrelated. The Arabidopsis (Arabidopsis thaliana) MUR10 gene is required for normal primary cell wall carbohydrate composition in mature leaves as well as for normal plant growth, hypocotyl strength, and fertility. The overall sugar composition of young mur10 seedlings is not significantly altered; however, the relative proportion of pectin side chains is shifted toward an increase in 1 → 5-α-arabinan relative to 1 → 4-β-galactan. mur10 seedlings display reduced fucogalactosylation of tightly cell wall-bound xyloglucan. Expression levels of genes encoding either nucleotide sugar interconversion enzymes or glycosyl transferases, known to be involved in primary and secondary cell wall biosynthesis, are generally unaffected; however, the CesA7 transcript is specifically suppressed in the mur10-1 allele. The MUR10 locus is identical with the CesA7 gene, which encodes a cellulose catalytic subunit previously thought to be specifically involved in secondary cell wall formation. The xylem vessels in young mur10 hypocotyls are collapsed and their birefringence is lost. Moreover, a fucogalactosylated xyloglucan epitope is reduced and a 1 → 5-α-arabinan epitope increased in every cell type in mur10 hypocotyls, including cells that do not deposit secondary walls. mur10 also displays altered distribution of an arabinogalactan-protein epitope previously associated with xylem differentiation and secondary wall thickening. This work indicates the existence of a mechanism that senses secondary cell wall integrity and controls biosynthesis or structural remodeling of primary cell walls and cellular differentiation

A Transcriptional and Metabolic Framework for Secondary Wall Formation in Arabidopsis

Plant cell walls are essential for plant growth and development. The cell walls are traditionally divided into primary walls, which surround growing cells, and secondary walls, which provide structural support to certain cell types and promote their functions. While much information is available about the enzymes and components that contribute to the production of these two types of walls, much less is known about the transition from primary to secondary wall synthesis. To address this question, we made use of a transcription factor system in Arabidopsis (Arabidopsis thaliana) in which an overexpressed master secondary wall-inducing transcription factor, VASCULAR-RELATED NAC DOMAIN PROTEIN7, can be redirected into the nucleus by the addition of dexamethasone. We established the time frame during which primary wall synthesis changed into secondary wall production in dexamethasone-treated seedlings and measured transcript and metabolite abundance at eight time points after induction. Using cluster- and network-based analyses, we integrated the data sets to explore coordination between transcripts, metabolites, and the combination of the two across the time points. We provide the raw data as well as a range of network-based analyses. These data reveal links between hormone signaling and metabolic processes during the formation of secondary walls and provide a framework toward a deeper understanding of how primary walls transition into secondary walls

Hypocotyl lengths and cross sections of samples tested with experimental setup 1 (horizontal test, water vapour).

<p>For each series n > 18 replicas were analyzed. Error bars depict standard deviation.</p

List of genes encoding the selected TFs.

<p>The genes in Table 1 were selected on the basis of co-expression in gene vicinity networks for <i>MYB46</i> and <i>CESA7</i>, <i>CESA8</i> and <i>CESA4</i>. Locus, common name, T-DNA insertion line used for functional studies, and description of structural domains or type of DNA binding domain found in each TF are indicated. T-DNA insertion mutants in the selected group of genes were further characterized in their response towards <i>B</i>. <i>cinerea</i> and <i>P</i>. <i>cucumerina</i>.</p><p>List of genes encoding the selected TFs.</p

Characterization of <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i> and <i>at1g66810</i> mutants.

<p>(<b>A</b>) Oregon Green 488 dextran-derived fluorescence upon infiltration of the full expanded leaves with the apoplastic pH indicator. In comparison to Col-0, more intense fluorescence emission was observed in the four mutants, and was particularly intense in <i>pap2</i> plants, indicative of enhanced alkalinization of the apoplast in mutants compared to Col-0 plants. (<b>B</b>) Estimation of plasma membrane-anchored (GPI-AGP) and free AGP content in Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> insertion mutant plants using β-d-Glucosyl Yariv reagent. AGP content was calculated with respect to a regression curve obtained by a radial diffusion assay in agarose plates containing Yariv regent and increasing amounts of gum Arabic (calibrating curve on the left). (<b>C</b>) Scanning electron microscopy (SEM) of leaf epidermis in Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> mutants. Only <i>pap2</i> exhibited a slight increase in epidermal cell size which was variable among different leaves of different plants. (<b>D</b>) Histochemical detection of lignin in proximal stem sections of Col-0 and <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i> and <i>at1g66810</i> mutant stems. Stem sections were stained with phloroglucinol-HCl (red color) for lignin detection in the interfascicular fiber walls and xylem cells, as observed with light microscopy. if, Interfascicular fibers; pi, Pith parenchyma; x, Xylem. (<b>E-F</b>) Cotyledon vein patterns were altered in <i>zfp2</i>, <i>bhlh99</i>, <i>pap2</i>, and <i>at1g66810</i> insertion mutants. <b>E</b>, magnified pictures showing pattern defects exhibited by some mutants (e.g., 2 loops; right picture) compared to the most common 4 loop pattern observed in Col-0 (left picture). <b>F</b>, columns 2–7: number of cotyledons displaying the venation pattern depicted at the top, which ranged from the most common 4 loops observed in Col-0 to the less common phenotype of 2+1 or 2 loops, but prevalent in the <i>pap2</i>, <i>bhlh99</i>, or <i>zfp2</i> mutants. “n” = total number of scored cotyledons.</p

Age Effects on Hypocotyl Mechanics - Fig 6

<p>Comparison of the results of the micromechanical tests with samples tested submerged in an osmotic liquid (green, Setup 2) and in air, kept in the native state by a humidifier (blue, Setup 1) for tensile stiffness (A) and fracture stress (B). The change in diameter from the initial value before the tensing to the point of maximal stress in the submerged setup (C).</p