Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.     

A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.     

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.     

Enhancement of the citrus immune system provides effective resistance against Alternaria brown spot disease

In addition to basal defense mechanisms, plants are able to develop enhanced defense mechanisms such as induced resistance (IR) upon appropriate stimulation. We recently described the means by which several carboxylic acids protect Arabidopsis and tomato plants against fungi. In this work, we demonstrate the effectiveness of hexanoic acid (Hx) in the control of Alternaria brown spot (ABS) disease via enhancement of the immune system of Fortune mandarin.     

Control of the phytopathogen Botrytis cinerea using adipic acid monoethyl ester

The in vitro and in vivo antifungal activity of adipic acid monoethyl ester (AAME) on the necrotrophic pathogen Botrytis cinerea has been studied. This chemical effectively controlled this important phytopathogen, inhibited spore germination and mycelium development at non-phytotoxic concentrations. The effectiveness of AAME treatment is concentration-dependent and influenced by pH. Spore germination in the presence of AAME is stopped at a very early stage, preventing germ tube development. In addition, cytological changes such as retraction of the conidial cytoplasm in the fungus are observed. AAME was also found to act on membrane integrity, affecting permeability without exhibiting lytic activity, as described previously for other antifungal compounds. Polyamine content in the mycelium of B. cinerea was also affected in response to AAME treatment, resulting in putrescine reduction and spermine accumulation similar to a number of antifungal agents. Microscopic observation of treated conidia after inoculation on tomato leaves suggested that inhibited spores are not able to attach to and penetrate the leaf. Finally, AAME completely suppressed the grey mould disease of tomato fruits under controlled inoculation conditions, providing evidence for its efficacy in a biological context and for the potential use of this chemical as an alternative fungicide treatment.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Absence of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces susceptibility to Botrytis cinerea in tomato

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.