Mycobacterium smegmatis cell fractionation was carried out essent

Mycobacterium smegmatis cell fractionation was carried out essentially as described earlier, with minor modifications (Delogu et al., 2004). Briefly, the recombinants grown up to the late log phase were harvested by centrifugation at 3000 g for 10 min at 4 °C, followed by washing with cold phosphate-buffered saline (PBS) and finally sonication in PBS containing the protease inhibitor P-8849 (Sigma-Aldrich). The whole-cell lysate thus prepared was centrifuged at 20 000 g to separate the insoluble (pellet) and the soluble (supernatant) fractions. Samples were subjected to SDS-PAGE as described by Laemmli (1970)

and subjected to Western blot analysis essentially as described earlier (Alone et al., 2007) using anti-GFP monoclonal antibody (Roche, Germay). The blot was developed with a horseradish peroxidase-labeled anti-mouse IgG

antibody (Sigma-Aldrich) and a chemiluminescent buy 3-Methyladenine substrate system (Biological Venetoclax supplier Industries, Israel). Mycobacterium smegmatis cells were allowed to grow at 200 r.p.m. at 37 °C. OD600 nm was measured every 3 h using a Perkin-Elmer spectrophotometer. To analyze the growth kinetics, OD600 nm was plotted against time on a semi-log plot. Wild-type and transformed M. smegmatis were fixed with 4% paraformaldehyde and coated on poly-lysine treated coverslips, which were then mounted on slides using vectashield mounting medium (Vector Laboratories Inc.). Microscopic visualization was performed on click here a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) using an oil immersion objective. Immunoelectron microscopy of M. smegmatis

cells was performed essentially as described earlier (Burghardt & Droleskey, 2006) at the Electron Microscopic Facility, Advanced Instrument Research Facility, JNU, New Delhi. Briefly, M. smegmatis cells from log-phase cultures were fixed with 4% paraformaldehyde containing 0.5% glutaraldehyde and concentrated in 2% agar. The agar-encased bacteria were then dehydrated and embedded using LR white resin (Electron Microscopy Sciences). Thin sections (100 nm) were obtained using Leica Ultracut (Leica, Germany) and processed for immunostaining using anti-GFP antibody and gold (10 nm)-labeled anti-mouse secondary antibody. Unrelated antibody was used at a similar dilution as a negative control. The immunostained sections were viewed using a Jeol 2100F transmission electron microscope (Jeol Analytic Instruments). The colonies of the M. smegmatis transformed with pVV1651cGFP (pVV1651cGFPMs) appeared on the solid agar-based medium after 4 days of plating, while the colonies transformed with vector alone (pVVGFPMs) appeared within 3 days. On day 5, PE_PGRS30-transformed M. smegmatis colonies were smaller in size when compared with the control (Fig. 1a and b). The M. smegmatis cultures transformed with the pVV1651c showed a significant lag in growth when compared with that transformed with the pVV16.

In order to have an accurate assessment of task performance in th

In order to have an accurate assessment of task performance in the fMRI environment, the timing of the stimulus and response mode of the RGS were adapted in accordance with the fMRI scanning requirements and timings (Fig. 2). Subjects were presented with image sequences generated by the VR machine, showing the arms of an avatar in a green landscape following the standard RGS protocol. Colored balls moving at various speeds and angles relative to the subject approached the avatar in the right or left visual field from the horizon in a first or third person perspective (Fig. 1). When a ball approached a virtual hand, the subjects had to press a button

with the index finger of their corresponding right or left hand. The time window for successfully catching the ball was 1000 ms (500 ms before and 500 s after crossing BMS-777607 the flight direction of the ball and the path of the catching hand). This was chosen to account for the fact that, in the RGS, the avatar’s position is fixed, whereas in real life

one would be able to move one’s body forwards or backwards in order to catch a flying ball. When the ball was missed, it passed by and left the field of view. When the ball was caught, the subjects could view the caught ball for the subsequent 8 s to let the hemodynamic Protein Tyrosine Kinase inhibitor response return to baseline. After a short blank display of the landscape, the next trial

began with a reappearance of the avatar. There were 24 repetitions of each trial, and each trial lasted 24 s. In a mixed event-related experimental design, subjects were presented with three different experimental conditions in separate Benzatropine scanning sessions in a pseudo-random order (Fig. 2): (i) action condition – the subjects were required to actively catch the balls by pressing the corresponding button (left/right) with their index finger; (ii) observation condition – the subjects were required to observe the avatar catching the balls; and (iii) imagination condition – the balls disappeared during their flight towards the avatar, and the subjects were required to imagine catching the ball at the right moment; for balls on the right, they had to indicate this by a right button press, and vice versa. Passive viewing of the landscape served as the baseline. Behavioral data were analysed with spss software (Version 20; IBM, Armonk, NY, USA). Prior to statistical analysis, data were tested for normal distribution with the Kolmogorov–Smirnov test. In case of a deviation from normal distribution, median scores were calculated, and the non-parametric Wilcoxon test was used to compare data (corrected α = 0.008). Imaging data were analysed with the brainvoyager qx software package (Brain Innovation, Maastricht, the Netherlands).

, 2010), it provides a broader view of fixed samples, aiding in c

, 2010), it provides a broader view of fixed samples, aiding in comparative fluorescent channel intensity calculations. As would be expected, the guinea-pig antibody against whole C. burnetii produced a strong fluorescent signal. Changes in the IcmT levels were measured against this standard. Fluorescence was observed in both channels for each time point sampled (data not shown). Figure 4c shows a graphical representation of these data relative to 0 hpi. Analysis

revealed that from 0 to 24 hpi, a significant increase (P<0.05) in the amount of IcmT relative to whole C. burnetii occurred between each time point measured. From 24 to 168 hpi, a statistically significant change was not detected. Although subtle, click here these data demonstrate SCH772984 purchase that IcmT expression increases early during infection, and then remains relatively unchanged for the duration of the infectious cycle. Whether these changes during this crucial time in PV development are required for C. burnetii survival is yet to be determined. However, the need for secreted effector proteins to control the development and trafficking of the PV early during the infectious cycle would likely be central to C. burnetii’s survival. Whether the IcmT detected at 0 hpi is part of a functional T4BSS

structure poised to secrete effector proteins upon host cell contact or whether a functional T4BSS structure is assembled once the bacteria enter the host cell

remains to be elucidated. Combined with our RT-qPCR analysis, these data suggest that C. burnetii T4BSS IcmT expression closely follows the increase in icmT transcript early during infection (0–24 hpi) and becomes relatively uniform for the duration of the infection (24–168 hpi). A comparison of Fig. 3 (icmT) and Fig. 4c indicates that an increase in RNA expression early during infection is followed by an increase in IcmT protein levels from a low at 0 hpi. Although the increase in RNA is modest, the relationship between the RNA and the corresponding IcmT protein expression indicates that temporal regulation of IcmT  expression exists during the C. burnetii infectious cycle. In summary, we have shown that the C. burnetii T4BSS RI is expressed as a set of three operons and that de novo transcription and translation of C. burnetii T4BSS HAS1 genes is present as early as 8 hpi. In addition, we have shown that an increase in transcription is accompanied by an increase in at least one protein, IcmT, in the first 24 hpi. Protein levels for the C. burnetii T4BSS RI IcmT homolog appear to be relatively constant at later stages of an infection (48–168 hpi). These data provide an increase in our understanding of the temporal regulation of the C. burnetii T4BSS early during infection and indicate that this bacterial virulence mechanism is maintained throughout infection.

bethesdensis (AY788950), A cryptum (D30773), Swaminathania salit

bethesdensis (AY788950), A. cryptum (D30773), Swaminathania salitolerans (AB445099), Saccharibacter floricola (NR_024819), and Neoasaia chiangmaiensis (AB208549), were obtained from the NCBI website at To construct the phylogenetic tree of AAB, these 37 sequences were collected and nucleotide sequence alignment was carried out using clustalw (Larkin et al., 2007). We selleck chemicals llc used the mega version 4.0 package

to generate phylogenetic trees to study the phylogenetic relationship based on 16S rRNA gene with the neighbor-joining (NJ) approach and 1000 bootstrap replicates (Tamura et al., 2007). Three hundred and ninety-one unique complete microbial genome sequences (one genome per genus) were obtained from the NCBI FTP website at Only amino acid-coding sequences on the chromosomes were used

for comparative analysis. For a homology search, a dataset of all proteins was constructed. The dataset of all proteins was constructed from all amino acid sequences from 391 complete microbial genomes. Four hundred and forty-three proteins on the KEGG metabolic map of G. oxydans were used as a query for the blastp homology search against the dataset of all proteins (Altschul et al., 1997; Kanehisa, 1997; Ogata et al., 1999; Kanehisa & Goto, 2000; click here Kanehisa et al., 2002, 2004, 2006, 2008, 2010). Of the 443 proteins, 293 were selected for further analysis because these ORFs exist in all three genera, Gluconobacter, Gluconacetobacter, and Acetobacter. Each homolog was identified by a homology search of amino acid sequence using the blastp filtering

expectation value of e-value ≤10−10 and sequence overlap ≥70% (Altschul et al., 1997). The top 50 hits were collected and multifasta files were created for phylogenetic analysis using house-written ruby scripts. The previously published complete genome sequences Pyruvate dehydrogenase of Acetobactericeae, G. oxydans, G. diazotrophicus, A. pasteurianus, G. bethesdensis, and A. cryptum were obtained from the NCBI FTP website at (Prust et al., 2005; Greenberg et al., 2007; Azuma et al., 2009; Bertalan et al., 2009). Only protein-coding genes on the chromosomes were used for the identification of orthologous groups. Each orthologous gene was identified by homology searches for amino acid sequence using the blastp filtering expectation value of e-value ≤10−10 and sequence overlap ≥70% (Altschul et al., 1997). All ORFs were searched against each species, and the reciprocal best hits were regarded as being orthologous genes. If genes were orthologous among all species present, the group was defined as a unique orthologous dataset.

Infection of mice with this mutant strain demonstrated blocking α

Infection of mice with this mutant strain demonstrated blocking α-glucan synthesis has no effect on G217B virulence (Edwards et al., 2011). Analysis of a G217B strain in which α-glucan synthesis was independently blocked by RNAi showed a similar lack of requirement for α-glucan in G217B intramacrophage replication and in lung infection. Interestingly,

although G217B yeast cells lack α-glucan, they can still prevent Dectin-1 recognition of cell wall β-glucan (Edwards et al., 2011). The growth stage-dependent mechanism by which G217B yeast accomplish this is unknown. Thus, G217B (representing chemotype I) and G186A (representing the chemotype II lineages) significantly differ in their mechanisms of pathogenesis with regard to yeast cell wall glucans and avoidance of detection by host immune cells. Yps3 is a secreted cell wall factor with sequence homology to the B. dermatitidis adhesin BAD1. Similar to BAD1, the Yps3 protein AZD5363 interacts

ABT-888 in vivo with chitin on the G217B yeast cell wall (Bohse & Woods, 2005). G217B yeast in which Yps3 production is blocked by RNAi grow similar to the wild-type strain in vitro and exhibit similar virulence in macrophages. However, the Yps3-deficient strain is defective in dissemination to the spleen and liver, implicating Yps3 in progression toward disseminated disease (Bohse & Woods, 2007a). Although the YPS3 gene is transcribed transiently by G186A strains upon shift from 25 to 37 °C, expression is not maintained in the yeast phase (Keath et al., 1989). Sustained expression of the gene and production of the Yps3 protein is restricted to NAm2 strains such as G217B, in vitro (Bohse & Woods, 2007b). Yps3 production in vivo remains to be tested for all Histoplasma strains. In addition, the YPS3 genes of different strains encode proteins with variable numbers of tandem repeats (two in NAm2, 11–12 in Panamanian strains, and 18–20 in NAm1). Thus, both structural and regulatory differences exist among the strains with regards to Yps3.

No genetic tests have been performed to test whether G186A virulence requires Yps3, but the lack of Yps3 production by G186A suggests Clomifene that Yps3 represents a distinct pathogenic mechanism for NAm2 strains. Histoplasma yeast are sensitive to the availability of iron and expresses factors to acquire sufficient iron from the environment. Iron restriction by the host is an important mechanism for restriction of Histoplasma yeast growth similar to control of other intracellular pathogens (Newman et al., 1994). Histoplasma yeast require iron for both in vitro growth (Timmerman & Woods, 1999, 2001) and growth in macrophages (Lane et al., 1991; Newman et al., 1994, 1995). Genetic studies have identified the several gene products as important mechanisms for Histoplasma iron acquisition (Hwang et al., 2003; Hilty et al., 2008, 2011; Zarnowski et al., 2008). Of these genes, only SID1 has been depleted in both G217B and G186A strains (Hwang et al., 2003; Hilty et al.

There are limited long-term safety data regarding its use with as

There are limited long-term safety data regarding its use with aspirin, clopidogrel or warfarin. Cilostazol is contraindicated in patients with congestive cardiac failure, previous ventricular arrhythmias and prolonged QT, and those with significant bleeding history. It should be avoided in moderate to

severe hepatic dysfunction and renal dysfunction (eGFR less than 25ml/min/1.73m2). Frequently encountered adverse effects leading to discontinuation of the drug include headache, palpitations and diarrhoea. Other significant side effects GSI-IX mw include thrombo-cytopenia, agranulocytosis, cardiac disorders and allergic reactions. Since 1998 several randomised controlled trials have been published assessing the therapeutic use of cilostazol Ivacaftor nmr for intermittent claudication. One of the largest initial multicentre, randomised, doubled-blinded

trials, conducted by Beebe et al.,1 compared cilostazol with placebo. The study included 516 men and women aged over 40 years with moderately severe chronic intermittent claudication. Patients were randomised to receive cilostazol 100mg, cilostazol 50mg or placebo twice daily for 24 weeks. Outcome measures included walking distances using treadmill testing, quality of life measures and cardiovascular and all-cause mortality. Improved walking distances were observed as early as four weeks in both cilostazol groups compared with placebo. The cilostazol 100mg twice daily group (n=138) had the greatest benefit at 24 weeks; the pain-free walking distance increased from 70.4m to 137.9m, a 59% geometric mean improvement, compared to 20% in the placebo group (p<0.001) and the maximal walking distance increased from 129.7m to 258.8m. A meta-analysis2 of eight randomised, placebo-controlled trials of cilostazol for intermittent claudication included 2702 patients with stable, moderate to severe intermittent claudication over 12 to 24 week trial periods. Similarly, cilostazol 100mg twice daily was found

to significantly improve pain-free walking distance by 67% and maximal walking distance by 50% (p<0.05). Subgroup analysis for gender, age and diabetes found no differences. Decitabine nmr Two studies included comparison to another therapeutic agent available for intermittent claudication, pentoxifylline, and found it to be comparable to placebo. The same two studies also measured plasma lipids and found that cilostazol 100mg twice daily increased HDL cholesterol by 12.8% and decreased triglycerides by 15.8% at 24 weeks; this was significant when compared to placebo and pentoxifylline. Initial studies were not powered to detect significant efficacy in the population with diabetes. Another meta-analysis3 examined eight phase III trials looking specifically at the use of cilostazol 100mg twice daily compared to placebo in diabetic and non-diabetic patients.

Genes involved in cysteine metabolism are important for tellurite

Genes involved in cysteine metabolism are important for tellurite resistance in bacteria (Chasteen et al., 2009). We then decided to compare the tellurite sensitivity of strains BSIP1215 and BSIP1793 (ΔcymR). On plates containing methionine, the ΔcymR mutant was less resistant to tellurite than the wild-type strain with a growth inhibition area diameter of 47.7 and 30.3 mm, respectively (Fig. 4b). In contrast, on plates containing cystine, the same growth inhibition area diameter was obtained for both strains (40.2 mm for BSIP1793 and 40.6 mm for BSIP1215) (Fig. 4b). In addition, the black deposits were much more prevalent for the ΔcymR mutant than for

the wild-type strain and the selleck products blackening mostly surrounded the paper disk for strain BSIP1793

(Fig. 4a, left Y-27632 in vitro panel). Tellurite might be reduced by the H2S produced by bacteria. The significant amount of H2S produced in the ΔcymR mutant was probably responsible for the quantity of tellurium deposits observed with this mutant. The diffusion of H2S into the plate could also explain why tellurite reduction occurred even in the zone of growth inhibition. To confirm the possible role of H2S in this phenomenon, we repeated the same disk assay, but kept the lid of the plate open in a moisturized atmosphere, allowing H2S diffusion outside from the plate. The growth inhibition area diameter of the ΔcymR mutant then markedly increased in the open plates, reaching 52.7 mm instead of 38.1 mm for the wild-type strain. Simultaneously, the blackening around the paper disk disappeared

selleck kinase inhibitor (Fig. 4a, right panel). A similar result was obtained when 5 mL of alkaline agar enriched with zinc acetate was poured on the lid to absorb H2S (data not shown). This indicated that H2S obtained from cysteine degradation probably participated in tellurite reduction, protecting the ΔcymR mutant from its toxicity. When H2S escaped from the plate, we observed a drastic increase in tellurite sensitivity for the ΔcymR strain similar to that obtained in the presence of methionine under conditions producing less H2S (Fig. 3a). We then tested the effect of CymR inactivation on the susceptibility of B. subtilis to other stress stimuli. We compared the sensitivity of strains BSIP1215 and BSIP1793 (ΔcymR) to paraquat, H2O2 and diamide using disk diffusion assays. The ΔcymR mutant was significantly more sensitive than the wild-type strain to diamide, a specific thiol oxidant that causes disulfide stress. This effect was observed with plates containing cystine or methionine (Table 1, data not shown). We further tested the effect of H2O2 and paraquat. On plates with methionine, the growth inhibition area in the presence of 10 μL of 2 M paraquat was 58.8 mm for the ΔcymR mutant and 49.3 mm for the wild-type strain. Under the same conditions, the zone of growth inhibition in the presence of 10 μL of 10 M H2O2 was 52.1 mm for the ΔcymR mutant and 41.4 mm for the wild-type strain.

2A; F1,27 = 5856,

P < 001, ηρ2 = 068) The main effect

2A; F1,27 = 58.56,

P < 0.01, ηρ2 = 0.68). The main effect of temporal attention (time expectation) was also significant (Fig. 2B; F1,27 = 5.20, P = 0.03, ηρ2 = 0.16), with overall faster responses at the expected time point. Importantly, we found a significant interaction between modality prevalence and time expectation (Fig. 2C; F1,27 = 17,85, click here P < 0.01, ηρ2 = 0.39). While participants reacted significantly faster to primary targets presented at the expected, and overall more likely, time point compared to the unexpected time point (t28 = −3.75, P < 0.01), we found the reverse, nearly significant, pattern for targets in the secondary modality (slower RTs at expected vs. unexpected time point; t28 = 1.77, P = 0.09). This reveals a breach in cross-modal synergy and suggests, instead, a decoupling of time expectation across

modalities. This decoupling was qualified by the significant triple interaction between interval, modality prevalence and expected time point (F1,27 = 7.32, P = 0.01, ηρ2 = 0.21), suggesting different patterns for the early and late time points (see Fig. 2D and E). In order to follow up on this interaction, we ran separate anovas for each (early and late) interval. Both time intervals revealed an interaction between modality prevalence and temporal expectation, just as in the main (pooled) data analysis. For the primary modality targets, time expectancy effects (faster RTs when the time point was the expected Gemcitabine manufacturer than the unexpected one) were significant at the early time point (1 s; t28 = −2.51, P = 0.02) as well as for the late (2.5 s) time point (t28 = −2.42, P = 0.02). In the case of the LBH589 secondary modality, however, this tendency levelled off (t28 = −0.79, P = 0.43) in the early time point and was completely reversed in the second time point. That is, responses to targets in the secondary modality were significantly slower if participants expected a target in the primary modality in that interval, compared to the unexpected interval

(t28 = 2.71, P = 0.01). In summary, upon targets appearing after 1 s, the secondary modality did not follow the expectation effects of the primary modality. Furthermore, upon targets appearing after 2.5 s, we found expectancy effects to abide by the relative likelihood of the secondary modality and run counter to the likelihoods of the primary modality. This pattern was equivalent for the two combinations of primary/secondary modalities (vision/touch, or touch/vision), as the interaction between primary modality, modality prevalence, expected time point and onset time did not reach statistical significance (t28 = 1.95, P = 0.17, ηρ2 = 0.07). However, for the sake of confirmation, we decided to run statistics on each modality combination separately. When touch was the primary modality, participants responded significantly faster to tactile targets if they were presented at the expected than at the unexpected time point (t13 = −4.26, P < 0.01).

, 2010) To confirm that this advantage applies to Purkinje cells

, 2010). To confirm that this advantage applies to Purkinje cells, we sought to molecularly perturb their early developmental processes by IUE. The ataxic mouse mutant staggerer is caused by a deletion in the gene encoding RORα1 (Sidman et al., 1962; Hamilton et al.,

1996). As RORα1 lacking Ixazomib chemical structure the putative ligand-binding domain (RORα1DN) serves as a dominant-negative mutant in cultured muscle cells (Lau et al., 1999, 2004) (Fig. 5A), we introduced two plasmids, pCAG-RORα1DN-HA, in which HA-tagged RORα1DN was placed under the CAG promoter, and pCAG-EGFP, into Purkinje cells by IUE at E11.5. The mice were fixed at P9, and sagittal sections at the vermis were immunostained for calbindin and HA to visualize Purkinje cells and RORα1DN, respectively.

Confocal microscopy showed that almost all the control calbindin-positive Purkinje cells expressing EGFP had single primary dendrites (96.2%, 102 of 106 cells; Fig. 5B and C). By contrast, only half of the calbindin-positive Purkinje cells expressing EGFP and RORα1DN-HA had a single primary dendrite (49.5%, 50 of 101 cells; P < 0.0001 vs. control, χ2 test), and the remaining cells had from two to five primitive dendrites (Fig. 5B and C). Furthermore, while all the control Purkinje cells expressing EGFP were arranged in a monolayer together with non-transfected Purkinje cells, a small number of Purkinje cells expressing RORα1DN-HA (six of 101) were mislocalized to the granular layer (Fig. 5B, arrowheads). These phenotypes observed in Purkinje cells expressing RORα1DN-HA were reminiscent of those observed in staggerer Purkinje cells (Soha & Herrup, 1995; Nakagawa et al., 1998). These results clearly indicate that certain

staggerer phenotypes can be mimicked by the IUE-mediated expression of dominant-negative RORα1 in single Purkinje cells during early development. Although IUE has several advantages as a method for transferring genes into neurons in vivo, it has never been applied Ponatinib chemical structure to cerebellar Purkinje cells, key neurons for regulating cerebellar functions. In the present study, we showed that Purkinje cell progenitors at E11.5 could be most efficiently and preferentially transfected by IUE, by properly adjusting the angle and direction of the electrodes (Fig. 1). Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at P28 (Fig. 2). We also showed that simultaneous expression of three different fluorescent proteins (Fig. 4) and expression of a large gene (Bassoon; Fig. S4) could be successfully achieved by IUE in Purkinje cells. In addition, by using three plasmids encoding the L7 promoter and an inducible Cre/Lox system, we could achieve temporal and Purkinje-cell-specific transgene expression (Fig. 3).

The H2O2-induced transcript levels of most of the genes tested de

The H2O2-induced transcript levels of most of the genes tested depended strongly on ChAP1, and several required Skn7 for full induction. The gene for glutathione reductase (GLR1) was only twofold induced in Δchap1 compared with 52-fold in WT and 16-fold and Δskn7. In the double mutant, the transcript level was similar to the basal level in the untreated control, indicating that either transcription factor is sufficient only for partial expression, while both transcription factors are required for full expression (Fig. 2). TRX2 showed the same

pattern as GLR1, but the additive effect was not statistically significant. The TRR1 gene is under the regulation of ChAP1 alone. While superoxide dismutase (SOD1) expression is not strongly decreased by loss of either ChAP1 or Skn7 alone, the CX-4945 in vitro double mutant failed see more to upregulate the expression of SOD1. The catalase genes CAT1 and CAT3 seem ChAP1 dependent and Skn7 independent; however, this regulation is not significant at P < 0.01 by the multiple-comparison t-test used here. CAT2 is expressed in all three mutants. The expression of γ-glutamylcysteine

synthetase (GSH1) was also tested, and only minor upregulation was observed in WT and Δskn7. To test whether both ChAP1 and Skn7 contribute to virulence on the host, infection assays on maize were carried out. To inoculate undetached maize leaves, maize plants were grown in hydroponics (as described in the Materials and Methods section) for 12 days, the plants were removed from the medium and transferred into a tray where the roots were kept moist. Spores from Δchap1, Δskn7, Δchap1-Δskn7 (ΔΔ) and WT were prepared in ddW with 0.02% Tween 20; at least four plants were used for each mutant, and the second leaf was inoculated with three 7-μL droplets containing about 500 spores. Lesion areas were measured using imagej software from images taken 2 days after inoculation (Fig. 3a). Δchap1 and Δskn7 mutants were not significantly different in virulence from WT, whereas ΔΔ showed significantly smaller lesions (about 30% smaller, Fig. 3b). This demonstrates an additive contribution of the

two transcription factors that are lacking in the double mutant. These contributions may promote the ability D-malate dehydrogenase to counteract the plant’s oxidative burst as well as other stresses the pathogen encounters during infection. Thus, the double mutant may be sensitive to the HR or other plant defenses, preventing spreading of the mutant and resulting in smaller lesions than those formed by the WT. In vitro experiments showed that in response to some stressors, there is no additive contribution, whereas for others there is (Fig. 1). Loss of either of these transcription factors results in hypersensitivity to oxidants in plate assays, and the contribution of each is reflected in the expression of genes whose products allow the cell to cope with oxidative stress. ChAP1 is critical for increased expression of GLR1, TRR1, and TRX2 in response to hydrogen peroxide (Fig.