A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris

Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V(max,app) ranged from 10 to 900 U mg(-1), while the K(m,app) ranged from 0.16 to 0.6 mg ml(-1). Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.     

Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.Microbe-associated molecular patterns (MAMPs) are molecular signatures of entire groups of microbes and have key roles in activation of the defense response in plants (Jones and Dangl, 2006; Boller and Felix, 2009). Well-characterized proteinaceous MAMPs are bacterial flagellin, Elongation Factor Tu (EF-Tu), and Ax21, fungal xylanase, and oomycete pep13, an epitope of a secreted transglutaminase (Boller and Felix, 2009; Monaghan and Zipfel, 2012). Plants recognize MAMPs by means of pattern recognition receptors (PRRs), comprising a group of leucine-rich repeat (LRR) receptor-like kinases (RLKs) or LRR receptor-like proteins (RLPs) located in the plasma membrane (Greeff et al., 2012; Monaghan and Zipfel, 2012). The LRR-RLKs FLS2 and EFR recognize flg22 (the 22-amino acid eliciting epitope from the conserved flagellin domain) and elf18/elf26 (peptides derived from the N terminus of translation elongation factor EF-Tu), respectively (Gómez-Gómez and Boller, 2000; Kunze et al., 2004; Chinchilla et al., 2006; Zipfel et al., 2006). The fungal protein ethylene-inducing xylanase (EIX) is recognized by the tomato (Solanum lycopersicum) LRR-RLPs LeEIX1 and LeEIX2, of which only the latter mediates a necrotic response (Ron and Avni, 2004).BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) is an LRR-RLK acting as a common component in many RLK signaling complexes (Monaghan and Zipfel, 2012). Although it was originally identified as a protein that interacts with the brassinosteroid receptor BRI1 (Li et al., 2002; Nam and Li, 2002), BAK1 also forms ligand-induced complexes with FLS2 and EFR and contributes to disease resistance against the pathogens Pseudomonas syringae, Hyaloperonospora arabidopsidis (Hpa), and Phytophthora infestans (Chinchilla et al., 2007; Heese et al., 2007; Chaparro-Garcia et al., 2011; Roux et al., 2011). Tomato BAK1 interacts in a ligand-independent manner with LeEIX1 but not with LeEIX2, and the BAK1-LeEIX1 interaction is required for the ability of LeEIX1 to attenuate the signaling of LeEIX2 (Bar et al., 2010). BAK1 has also been shown to interact with another LRR-RLK, BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1). A bir1 mutant showed extensive cell death, activation of constitutive defense responses, and impairment in the activation of the mitogen-activated protein kinase MPK4 (Gao et al., 2009). sobir1 (for suppressor of BIR1) mutants suppress BIR1 phenotypes, and overexpression of SOBIR1 triggers cell death and defense responses (Gao et al., 2009). SOBIR1 does not physically interact with BIR1, suggesting that SOBIR1 mediates an alternative signal transduction route. A recent study showed that the tomato SOBIR1 interacts with RLPs and is required for RLP-mediated disease resistance (Liebrand et al., 2013).Endopolygalacturonases (PGs) are a class of pectinases that hydrolyze the homogalacturonan domain of pectic polysaccharides (van den Brink and de Vries, 2011). Secreted PGs are able to cause cell wall decomposition and tissue maceration and thereby act as virulence factors in several fungal pathogens, such as Aspergillus flavus, Claviceps purpurea, and Alternaria citri (Shieh et al., 1997; Isshiki et al., 2001; Oeser et al., 2002). The most extensively studied PGs from fungal plant pathogens are those of Botrytis cinerea (for review, see Zhang and van Kan, 2013b), a necrotrophic broad-host-range pathogen that contains six PG genes (designated Bcpg1–Bcpg6) in its genome (Wubben et al., 1999). Deletion of either Bcpg1 or Bcpg2 resulted in a strong reduction in virulence on tomato and broad bean (Vicia faba) leaves (ten Have et al., 1998; Kars et al., 2005), presumably because the enzymes have a detrimental effect on the integrity of host cell walls and tissues. Indeed, infiltrating BcPG2 into broad bean leaves or transient expression of BcPG2 in Nicotiana benthamiana led to tissue collapse and necrosis, and the necrotic response was abolished when the catalytic domain of the PG was mutated (Kars et al., 2005; Joubert et al., 2007). By contrast, Poinssot et al. (2003) reported that BcPG1 can activate plant defense responses in grapevine (Vitis vinifera) cell suspensions independently of its enzymatic activity, suggesting that the protein itself might be recognized by plant cells as an elicitor. These studies with seemingly opposing conclusions were conducted with different isozymes on distinct cell types of different plant species. Thus, it remained inconclusive whether plant responses observed after exposure to PGs are due to the structural damage resulting from pectin hydrolysis or to recognition of the protein as a MAMP (followed by downstream signaling responses).The degradation of pectin by PGs leads to the release of oligogalacturonides (OGAs), which may activate a variety of defense responses (Prade et al., 1999; D’Ovidio et al., 2004). OGAs act as damage-associated molecular patterns (DAMPs) via their perception by the cell wall-associated receptor Wall-Associated Kinase1 (WAK1; Brutus et al., 2010). Overexpression of WAK1 in Arabidopsis (Arabidopsis thaliana) enhances resistance to B. cinerea (Brutus et al., 2010).Here, we describe the occurrence of natural variation among Arabidopsis accessions in responsiveness to fungal PGs. Two accessions that strongly differed in their response to PGs were selected for further analysis. Cloning and functional characterization demonstrated that the gene RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1) encodes an LRR-RLP. Finally, we demonstrate that the LRR-RLK SUPPRESSOR OF BIR1 (SOBIR1) interacts with RBPG1 and is essential for responsiveness to fungal PGs.     

Epicardial Ablation: Prevention of Phrenic Nerve Damage by Pericardial Injection of Saline and the Use of a Steerable Sheath

Because of the close proximity of the phrenic nerve to the pericardium, phrenic nerve damage caused by epicardial ablation can easily occur. We report two cases of epicardial VT ablation where pericardial injection of saline, combined with the use of a steerable sheath, successfully prevents the phrenic nerve from being damaged.     

Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern

The objective of the present study was to investigate gene expression pattern of two docetaxel resistant MCF-7 breast carcinoma sublines step wisely selected in 30 and 120 nM docetaxel. Cell proliferation assay was performed in order to demonstrate development of docetaxel resistance. cDNA microarray analysis was performed using Affymetrix^® Human Genome U133 Plus 2.0 Arrays in duplicate experiments. Quantitative and semi-quantitative gene expression analysis was also performed to confirm gene expression analysis for selected genes. XTT results demonstrated that 30 (MCF-7/30nM DOC) and 120 nM (MCF-7/120nM DOC) docetaxel selected cells were 13- and 47-fold resistant, respectively. cDNA microarray analysis demonstrated that expression profiles of MCF-7 and MCF-7/30nM DOC were more similar to each other where expression profile of MCF-7/120nM DOC was different as examined by line graphs and scatter plots. 2,837 and 4,036 genes were significantly altered in 30 and 120 nM docetaxel resistant sublines, respectively. Among these, 849 genes were altered in common in two docetaxel resistant sublines. Antiapoptotic gene expression (e.g., Bcl-2 and APRIL) were noticeably altered in MCF-7/30nM DOC. However, docetaxel resistance in MCF-7/120nM DOC were more complicated with the involvement of ECM related gene expression, cytokine and growth factor signaling, ROS metabolism and EMT related gene expression together with higher level of MDR1 expression. Expression profiles in 30 and 120 nM docetaxel resistant sublines changed gradually with increasing resistance index. Drug resistance development seems to be step wise event in MCF-7 cells.     

Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum

The soil‐borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1‐like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine‐rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7_CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease‐controlling T3E, and sheds light on the co‐evolutionary arms race between the bacterium and its hosts.     

Thyroid Hormone Activates Brown Adipose Tissue and Increases Non-Shivering Thermogenesis - A Cohort Study in a Group of Thyroid Carcinoma Patients

Background/Objectives

Thyroid hormone receptors are present on brown adipose tissue (BAT), indicating a role for thyroid hormone in the regulation of BAT activation. The objective of this study was to examine the effect of thyroid hormone withdrawal followed by thyroid hormone in TSH-suppressive dosages, on energy expenditure and brown adipose tissue activity.

Subjects/Methods

This study was a longitudinal study in an academic center, with a follow-up period of 6 months. Ten patients with well-differentiated thyroid carcinoma eligible for surgical treatment and subsequent radioactive iodine ablation therapy were studied in a hypothyroid state after thyroidectomy and in a subclinical hyperthyroid state (TSH-suppression according to treatment protocol). Paired two-tailed t-tests and linear regression analyses were used.

Results

Basal metabolic rate (BMR) was significantly higher after treatment with synthetic thyroid hormone (levothyroxine) than in the hypothyroid state (BMR 3.8 ± 0.5 kJ/min versus 4.4 ± 0.6 kJ/min, P = 0.012), and non-shivering thermogenesis (NST) significantly increased from 15 ± 10% to 25 ± 6% (P = 0.009). Mean BAT activity was significantly higher in the subclinical hyperthyroid state than in the hypothyroid state (BAT standard uptake value (SUV^Mean) 4.0 ± 2.9 versus 2.4 ± 1.8, P = 0.039).

Conclusions

Our study shows that higher levels of thyroid hormone are associated with a higher level of cold-activated BAT.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02499471","term_id":"NCT02499471"}}NCT02499471     

Ecological correlates of species differences in the Lake Tanganyika crab radiation

The endemic crabs of Lake Tanganyika include a phenotypically diverse clade that exhibits recent divergence and low phylogenetic species resolution. There are indications that ecological niche segregation has played a prominent role in the divergence of this clade. We used habitat surveys, gut content analyses and stable isotope analyses to test the extent to which morphological species are ecologically different. Our data show some interspecific segregation in depth, substrate type and mean stable isotope signatures. At the same time, a considerable level of ecological niche overlap is evident among species of Platythelphusa that coexist in rocky littoral habitats. We consider these results in the framework of adaptive radiation theory, and we discuss general ramifications for the maintenance of species diversity in Lake Tanganyika.