Validating the Strategic Deployment of Blackleg Resistance Gene Groups in Commercial Canola Fields on the Canadian Prairies.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus L.) production in western Canada. Crop scouting and extended crop rotation, along with the use of effective genetic resistance, have been key management practices available to mitigate the impact of the disease. In recent years, new pathogen races have reduced the effectiveness of some of the resistant cultivars deployed. Strategic deployment and rotation of major resistance (R) genes in cultivars have been used in France and Australia to help increase the longevity of blackleg resistance. Canada also introduced a grouping system in 2017 to identify blackleg R genes in canola cultivars. The main objective of this study was to examine and validate the concept of R gene deployment through monitoring the avirulence (Avr) profile of L. maculans population and disease levels in commercial canola fields within the Canadian prairies. Blackleg disease incidence and severity was collected from 146 cultivars from 53 sites across Manitoba, Saskatchewan, and Alberta in 2018 and 2019, and the results varied significantly between gene groups, which is likely influenced by the pathogen population. Isolates collected from spring and fall stubble residues were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm9, AvrLm10, AvrLm11, AvrLepR1, AvrLepR2, AvrLep3, and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR-based markers. The Simpson's evenness index was very low, due to two dominant L. maculans races (AvrLm2-4-5-6-7-10-11 and AvrLm2-5-6-7-10-11) representing 49% of the population, but diversity of the population was high from the 35 L. maculans races isolated in Manitoba. AvrLm6 and AvrLm11 were found in all 254 L. maculans isolates collected in Manitoba. Knowledge of the blackleg disease levels in relation to the R genes deployed, along with the L. maculans Avr profile, helps to measure the effectiveness of genetic resistance.

Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.

A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and ß-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa 'americensis'.

A Six-Year Investigation of the Dynamics of Avirulence Allele Profiles, Blackleg Incidence, and Mating Type Alleles of Leptosphaeria maculans Populations Associated with Canola Crops in Manitoba, Canada.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is one of the most economically important diseases of canola (Brassica napus, oilseed rape) worldwide. This study assessed incidence of blackleg, the avirulence allele, and mating type distributions of L. maculans isolates collected in commercial canola fields in Manitoba, Canada, from 2010 to 2015. A total of 956 L. maculans isolates were collected from 2010 to 2015 to determine the presence of 12 avirulence alleles using differential canola cultivars and/or PCR assays specific for each avirulence allele. AvrLm2, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm11, and AvrLmS were detected at frequencies ranging from 97 to 33%, where the AvrLm1, AvrLm3, AvrLm9, AvrLepR1, and AvrLepR2 alleles were the least abundant. When the race structure was examined, a total of 170 races were identified among the 956 isolates, with three major races, AvrLm-2-4-5-6-7-11, AvrLm-2-4-5-6-7-11-S, and Avr-1-4-5-6-7-11-(S) accounting for 15, 10, and 6% of the total fungal population, respectively. The distribution of the mating type alleles (MAT1-1 and MAT1-2) indicated that sexual reproduction was not inhibited in any of the nine Manitoba regions in any of the years L. maculans isolates were collected.

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects.

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber-Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe(3+) and Cu(2+), using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu(2+)/ascorbate elicited more oxygen consumption than Fe(3+)/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu(2+)/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe(3+)/ascorbate. Finally, Fe(3+)/ascorbate and Cu(2+)/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu(2+) decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe(3+)/ascorbate and Cu(2+)/ascorbate but not by Cu(2+) alone. N-Acetylcysteine completely prevented the damage elicited by Cu(2+)/ascorbate, Fe(3+)/ascorbate and Cu(2+) alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe(3+)/ascorbate, partially reversed that of Cu(2+)/ascorbate but failed to reverse the damage promoted by Cu(2+) alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.

Antioxidant properties of a dry product from Vitis vinifera seeds (Leucoselect) in rat liver endoplasmic reticulum / Propiedades antioxidantes de un producto seco de semillas de Vitis vinifera (Leucoselect) en retículo endoplásmico de hígado de rata

Leucoselect is a commercial dry product obtained from grape seeds and enriched in procyanidins, which display antioxidant activity in virtue to their ability to scavenge oxygen free radicals and to chelate transition metal ions. The hypoxanthine/xanthine oxidase and Cu2+/ascorbate systems are capable of generating reactive oxygen species; the latter system can also promote non-specific binding of copper ions to proteins. Therefore, we assessed the ability of Leucoselect to inhibit oxidative phenomena elicited by both oxidative systems on rat liver microsomes: lipid peroxidation, oxidation of protein thiols, and inhibition of the cytochrome P450 system. The antioxidant activity of Leucoselect was a reflection of its ability to scavenge oxygen free radicals, chelate copper ions, and protect microsomal membranes through direct interaction. These mechanisms were displayed in a dependent manner with the type of biomolecule studied and also with the oxidative system employed, which is an interesting phenomenon to consider when evaluating the antioxidant activity of herbal products.

Leucoselect es un producto comercial seco obtenido de semillas de uva y enriquecido en procianidinas, las cuales presentan actividad antioxidante debido a su capacidad para atrapar radicales libres y quelar metales de transición. Los sistemas hipoxantina/xantina oxidasa y Cu2+/ascorbato generan especies reactivas del oxígeno; este último sistema también promueve la unión inespecífica de iones cobre a proteínas. Por lo tanto, evaluamos la capacidad de Leucoselect para inhibir los fenómenos oxidativos producidos por ambos sistemas oxidantes en microsomas hepáticos de rata: lipoperoxidación, oxidación de tioles proteicos e inhibición de la actividad del sistema citocromo P450. La actividad antioxidante de Leucoselect fue un reflejo de su capacidad de atrapar radicales libres del oxígeno, quelar iones cobre y proteger membranas microsómicas por interacción directa. Dichos mecanismos se manifestaron en forma dependiente del tipo de biomolécula estudiada y del sistema oxidante empleado, fenómeno interesante de considerar al evaluar la actividad antioxidante de preparados herbales.

Melatonin protects the cytochrome P450 system through a novel antioxidant mechanism.

Melatonin, an endogenous hormone, is used as an antioxidant drug in doses quite higher than the endogenous circulating levels of this hormone. Hepatic endoplasmic reticulum contains the cytochrome P450 (CYP450) system, which catalyzes one biotransformation pathway of melatonin; this organelle is also one of the main sources of reactive oxygen species in cells. Therefore, we proposed that the antioxidant activity of this hormone may have a biological relevance in the organelle where it is biotransformed. To evaluate this postulate, we used Fe(3+)/ascorbate, an oxygen free radical generating system that leads to lipid peroxidation, loss of protein-thiol content, and activation of UDP-glucuronyltransferase in rat liver microsomes. We found that mM concentrations of melatonin prevented all these oxidative phenomena. We also found that Fe(3+)/ascorbate leads to structural alterations in the CYP450 monooxygenase, the enzyme that binds the substrate in the CYP450 system catalytic cycle, probably through direct oxidation of the protein, and also inhibited p-nitroanisole O-demethylation, a reaction catalyzed by the CYP450 system. Notably, melatonin prevented both phenomena at microM concentrations. We provide evidence suggesting that melatonin may be oxidized by oxygen free radicals. Thus, we postulate that melatonin may be acting as an oxygen free radical scavenger, and Fe(3+)/ascorbate-modified melatonin would be directly protecting the CYP450 system through an additional specific mechanism. Pharmacological relevance of this phenomenon is discussed.

Comparative effects of superoxide anion and hydrogen peroxide on microsomal and cytosolic glutathione S-transferase activities of rat liver.

Glutathione S-transferases (GSTs) are isoenzymes occurring in the cytoplasm and as integral membrane proteins. In addition to their role in drug metabolism by conjugating electrophilic and lipophilic compounds with glutathione (GSH), these enzymes display multiple functions in cells, including antioxidant action. It has been generalized that reactive oxygen species (ROS) inhibit cytosolic GSTs and activate microsomal GSTs; some evidence shows, however, that different ROS-generating systems can inhibit microsomal GST activity. We therefore tested the effect of Fe3+/ascorbate, another ROS-generating system, on cytosolic and microsomal GST activities from rat liver cytosol and microsomes, respectively, and compared it to that of hydrogen peroxide (H2O2). We found that, while both agents displayed similar inhibitory effects on cytosolic GST activity, they promoted opposite effects on microsomal GST activity. Using specific antioxidant enzymes, we corroborated that the effect of Fe3+/ascorbate involves generation of O2(.-) without dismutation into H2O2. Since these ROS have physicochemical properties and redox potentials that are very distinct, their reactivity is different, and their oxidative action is likely to have different targets. We discuss how these properties are related with the oxidative potency of ROS, especially those of O2(.-) and H2O2.

RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice.

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Upregulation of gamma-glutamate-cysteine ligase as part of the long-term adaptation process to iron accumulation in neuronal SH-SY5Y cells.

Reactive iron is an important prooxidant factor, whereas GSH is a crucial component of a long-term adaptive system that allows cells to function during extended periods of high oxidative stress. In this work, the adaptive response of the GSH system to prolonged iron loads was characterized in human dopaminergic SH-SY5Y neuroblastoma cells. After the initial death of a substantial portion of the cell population, the surviving cells increased their GSH content by up to fivefold. This increase was traced to increased expression of the catalytic and modulatory subunits of gamma-glutamate-cysteine ligase. Under conditions of high iron load, cells maintained a low GSSG content through two mechanisms: 1) GSSG reductase-mediated recycling of GSSG to GSH and 2) multidrug resistant protein 1-mediated extrusion of GSSG. Increased GSH synthesis and low GSSG levels contributed to recover the cell reduction potential from -290 mV at the time of cell death to about -320 mV. These results highlight the fundamental role of GSH homeostasis in the antioxidant response to cellular iron accumulation and provide novel insights into the adaptive mechanisms of neurons subjected to increased iron loads, such as those observed in Parkinson's disease.

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms.

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.

Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1.

The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S -glutathionylation.

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Regulation of voltage-gated Ca2+ channels by calmodulin.

Calmodulin, a highly versatile and ubiquitously expressed Ca2+ sensor, regulates the function of many enzymes and ion channels. Both Ca2+-dependent inactivation and Ca2+-dependent facilitation of the voltage-gated Ca2+ channels Cav1.2 andCav2.1 are regulated through an interaction with Ca2+-bound calmodulin. This review addresses the functional regulation of Cav1.2 and Cav2.1 by calmodulin and discusses how Ca2+ binding to a single calmodulin molecule can regulate opposing functions of the voltage-gated Ca2+ channels.

Regulation of voltage-gated Ca2+ channels by calmodulin.

Calmodulin, a highly versatile and ubiquitously expressed Ca2+ sensor, regulates the function of many enzymes and ion channels. Both Ca2+-dependent inactivation and Ca2+-dependent facilitation of the voltage-gated Ca2+ channels Cav1.2 and Cav2.1 are regulated through an interaction with Ca2+-bound calmodulin. This review addresses the functional regulation of Cav1.2 and Cav2.1 by calmodulin and discusses how Ca2+ binding to a single calmodulin molecule can regulate opposing functions of the voltage-gated Ca2+ channels.