Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA‐Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.     

PsPMEP, a pollen-specific pectin methylesterase of pea (Pisum sativum L.)

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.     

Effect of dietary hempseed intake on cardiac ischemia-reperfusion injury

Polyunsaturated fatty acids (PUFAs) have significant, cardioprotective effects against ischemia. Hempseed contains a high proportion of the PUFAs linoleic acid (LA) and alpha-linolenic acid (ALA), which may have opposing effects on postischemic heart performance. There are no reported data concerning the cardiovascular effects of dietary hempseed intake. A group of 40 male Sprague-Dawley rats were distributed evenly into four groups that were fed for 12 wk a normal rat chow supplemented with hempseed (5% and 10%), palm oil (1%), or a 10% partially delipidated hempseed that served as a control. Plasma ALA and gamma-linolenic acid levels were significantly elevated in the rats that were fed a 5% or 10% hempseed-supplemented diet, but in heart tissue only ALA levels were significantly elevated in the rats fed these diets compared with control. After the dietary interventions were completed, postischemic heart performance was evaluated by measuring developed tension, resting tension, the rates of tension development and relaxation, and the number of extrasystoles. Hearts from rats fed a hempseed-supplemented diet exhibited significantly better postischemic recovery of maximal contractile function and enhanced rates of tension development and relaxation during reperfusion than hearts from the other groups. These hearts, however, were not protected from the occurrence of extrasystoles, nor were the increases in resting tension altered during ischemia or reperfusion as a function of any dietary intervention. Our data demonstrate that dietary hempseed can provide significant cardioprotective effects during postischemic reperfusion. This appears to be due to its highly enriched PUFA content.     

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.     

Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10‐100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000‐L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large‐scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1093–1102, 2014     

The clubroot pathogen Plasmodiophora brassicae: A profile update

Background

Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide.

Disease symptoms

In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients.

Host range

Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile.

Taxonomy

Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877).

Distribution

Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries.

Pathotyping

Based on its virulence on different hosts, P. brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P. brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts.

Effectors and resistance

After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date.

Important Link

Canola Council of Canada is constantly updating information about clubroot and P. brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/ .

Phytosanitary categorization

PLADBR: EPPO A2 list; Annex designation 9E.     

Biliary excretion of barium in the rat

Biliary excretion of barium was studied in Sprague-Dawley bile-duct-cannulated rats injected intravenously with 1.8 micrograms Ba/rat as 133Ba-labeled barium chloride. Approximately 0.5% of the barium dose was excreted into bile within 2 h. The time-course profile of biliary excretion of the radiotracer closely reflected that of plasma concentrations. Biliary barium levels reached their peak in the first 15-min period after administration and rapidly declined thereafter. The plasma-to-bile barium-concentration ratio was approx 1 at 2 h after injection. There was no tendency of barium to concentrate in liver, and the 133Ba levels in stomach and small intestine largely exceeded hepatic levels. There is evidence indicating that barium is predominantly excreted with feces following parenteral administration in rats and humans. The results of this study suggest that biliary excretion is of little quantitative importance and that physiological routes other than bile contribute to elimination of barium by the digestive tract.     

α2β1 Integrin Mediates Dermal Fibroblast Attachment to Type VII Collagen via a 158-Amino-Acid Segment of the NC1 Domain

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the α2 or β1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202–360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell–matrix interaction is mediated by the α2β1 integrin and is RGD independent.     

Environmental and biochemical trace-metal speciation studies by radiotracers and neutron activation analysis

Although knowledge of the total concentrations of trace metals in the environment and living organisms is still the essential starting point for any toxicological evaluation, it is, however, not sufficient to explain the mechanisms responsible for retention and toxic effect of trace metals. Differentiation between their chemical and biochemical forms is necessary. The need to resolve the total concentration of trace metals into single chemical species poses great experimental difficulties and imposes the use of very sensitive analytical techniques for trace metal determinations after specific preseparation procedures of the different chemical species. Thus, once the chemical or biochemical metal species have been selectively isolated, the speciation becomes merely an analytical problem, which requires high sensitivity and accuracy, as well as the evaluation of matrix effects, blanks, contamination, loss, and sampling. This paper describes the use of nuclear and radiochemical techniques related to chemical and biochemical speciation problems at the Joint Research Centre, Ispra, with carrier-free radiotracers and radiochemical neutron activation analysis.