Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute,Western X-disease phytoplasma,using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.     

The Effect of Chemical Information on the Spatial Distribution of Fruit Flies: I Model Results

Animal aggregation is a general phenomenon in ecological systems. Aggregations are generally considered as an evolutionary advantageous state in which members derive the benefits of protection and mate choice, balanced by the costs of limiting resources and competition. In insects, chemical information conveyance plays an important role in finding conspecifics and forming aggregations. In this study, we describe a spatio-temporal simulation model designed to explore and quantify the effects of these infochemicals, i.e., food odors and an aggregation pheromone, on the spatial distribution of a fruit fly (Drosophila melanogaster) population, where the lower and upper limit of local population size are controlled by an Allee effect and competition. We found that during the spatial expansion and strong growth of the population, the use of infochemicals had a positive effect on population size. The positive effects of reduced mortality at low population numbers outweighed the negative effects of increased mortality due to competition. At low resource densities, attraction toward infochemicals also had a positive effect on population size during recolonization of an area after a local population crash, by decreasing the mortality due to the Allee effect. However, when the whole area was colonized and the population was large, the negative effects of competition on population size were larger than the positive effects of the reduction in mortality due to the Allee effect. The use of infochemicals thus has mainly positive effects on population size and population persistence when the population is small and during the colonization of an area. Electronic Supplementary Material  The online version of this article () contains supplementary material, which is available to authorized users.     

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.     

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.