Dissecting Phaseolus vulgaris Innate Immune System against Colletotrichum lindemuthianum Infection

Background: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. Methodology and Principal Findings: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. Conclusions/Significance: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Mapping of angular leaf spot resistance QTL in common bean (Phaseolus vulgaris L.) under different environments

Background: Common bean (Phaseolus vulgaris L.) is the most important grain legume for human diet worldwide and the angular leaf spot (ALS) is one of the most devastating diseases of this crop, leading to yield losses as high as 80%. In an attempt to breed resistant cultivars, it is important to first understand the inheritance mode of resistance and to develop tools that could be used in assisted breeding. Therefore, the aim of this study was to identify quantitative trait loci (QTL) controlling resistance to ALS under natural infection conditions in the field and under inoculated conditions in the greenhouse. Results: QTL analyses were made using phenotypic data from 346 recombinant inbreed lines from the IAC-UNA x CAL 143 cross, gathered in three experiments, two of which were conducted in the field in different seasons and one in the greenhouse. Joint composite interval mapping analysis of QTL x environment interaction was performed. In all, seven QTLs were mapped on five linkage groups. Most of them, with the exception of two, were significant in all experiments. Among these, ALS10.1(DG,UC) presented major effects (R-2 between 16% - 22%). This QTL was found linked to the GATS11b marker of linkage group B10, which was consistently amplified across a set of common bean lines and was associated with the resistance. Four new QTLs were identified. Between them the ALS5.2 showed an important effect (9.4%) under inoculated conditions in the greenhouse. ALS4.2 was another major QTL, under natural infection in the field, explaining 10.8% of the variability for resistance reaction. The other QTLs showed minor effects on resistance. Conclusions: The results indicated a quantitative inheritance pattern of ALS resistance in the common bean line CAL 143. QTL x environment interactions were observed. Moreover, the major QTL identified on linkage group B10 could be important for bean breeding, as it was stable in all the environments. Thereby, the GATS11b marker is a potential tool for marker assisted selection for ALS resistance.

Cooking Effects on Iron and Proteins Content of Beans (Phaseolus Vulgaris L.) by GF AAS and MALDI-TOF MS

The effects of domestic cooking on proteins, organic compounds and Fe distribution in beans (Phaseolus vulgaris L.) were investigated. Sequential extraction with different extractant solutions (mixture of methanol and chloroform 1:2 v/v, water, 0.5 mol L-1 NaCl, 70% v/v ethanol and 0.5 mol L-1 NaOH) were used for extracting lipids, albumins, globulins, prolamins and glutelins, respectively. Iron determination by graphite furnace atomic absorption spectrometry (GF AAS), proteins by Bradford method and organic compounds by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were carried out in this work. High concentration of albumins, globulins and glutelins were found in raw beans, while in the cooked beans, albumins and glutelins are main proteins types. The MALDI-TOF MS spectra of raw and cooked beans revealed that the domestic cooking altered the molecular weight of the organic compounds, since that in the cooked beans were found compounds between 2 and 3.5 kDa, which were not presented in the raw beans. Besides this, in cooked beans were also observed the presence of four compounds of high molecular weight (12-16 kDa), being that in the raw grains there is only one (ca. 15.2 kDa). In raw grains is possible to observe that Fe is mainly associated to albumins, globulins and glutelins. For cooked grains, Fe is associated to albumins and globulins.

Cooking effects on iron and proteins content of beans (Phaseolus Vulgaris L.) by GF AAS and MALDI-TOF MS

The effects of domestic cooking on proteins, organic compounds and Fe distribution in beans (Phaseolus vulgaris L.) were investigated. Sequential extraction with different extractant solutions (mixture of methanol and chloroform 1:2 v/v, water, 0.5 mol L-1 NaCl, 70% v/v ethanol and 0.5 mol L-1 NaOH) were used for extracting lipids, albumins, globulins, prolamins and glutelins, respectively. Iron determination by graphite furnace atomic absorption spectrometry (GF AAS), proteins by Bradford method and organic compounds by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were carried out in this work. High concentration of albumins, globulins and glutelins were found in raw beans, while in the cooked beans, albumins and glutelins are main proteins types. The MALDI-TOF MS spectra of raw and cooked beans revealed that the domestic cooking altered the molecular weight of the organic compounds, since that in the cooked beans were found compounds between 2 and 3.5 kDa, which were not presented in the raw beans. Besides this, in cooked beans were also observed the presence of four compounds of high molecular weight (12-16 kDa), being that in the raw grains there is only one (ca. 15.2 kDa). In raw grains is possible to observe that Fe is mainly associated to albumins, globulins and glutelins. For cooked grains, Fe is associated to albumins and globulins.

Intraspecific competition in Zabrotes subfasciatus: Physiological and behavioral adaptations to different amounts of host

The effects of competition on populations of the bean weevil Zabrotes subfasciatus were analyzed during 41 generations under different competition levels. Three competition environments were established by maintaining the number of couples (6) and varying the amount of available host seeds: HC, high (limited availability of host: 1.35 g); IC, intermediate (intermediate availability of host: 6 g); and LC, low competition (abundance of host: 36 g). It was found that the distribution of the eggs laid on grains was different among treatments: in LC, for example, although females showed high fecundity (35.4 +/- 5.6 eggs/female) the number of eggs laid on each grain was small (1.2 +/- 0.4 eggs on each seed), thus avoiding larval competition of their offspring; whereas in HC treatment, females showed low fecundity (27.04 +/- 4.5 eggs/female) but laid many eggs on each grain (15.03 +/- 4.3 eggs). There were no changes in the ability to respond to different amounts of host via oviposition behavior (egg distribution) during 41 generations. However, HC females had more offspring than LC females under HC conditions. This suggests that HC insects evolved toward higher fitness in crowded conditions. In addition, after inverting the competition level, insects behaved independently of the treatment conditions they experienced through generations, thus showing that oviposition behavior is flexible. Taken together, our results show that Z. subfasciatus presents a broad range of behavioral and physiological responses which allows for quick and reversible adjustments to sudden changes in the amount of resources.

PHOSPHITE AS PHOSPHORUS SOURCE TO GRAIN YIELD OF COMMON BEAN PLANTS GROWN IN SOILS UNDER LOW OR ADEQUATE PHOSPHATE AVAILABILITY

The effects of foliar and soil applied phosphite on grain yield in common bean (Phaseolus vulgaris L.) grown in a weathered soil under low and adequate phosphate availability were evaluated. In the first experiment, treatments were composed of a 2 x 7 + 2 factorial scheme, with 2 soil P levels supplied as phosphate (40 e 200 mg P dm(-3) soil), 7 soil P levels supplied as phosphite (0-100 mg P dm(-3) soil), and 2 additional treatments (without P supply in soil, and all P supplied as phosphite). In the second experiment, treatments were composed of a 2 x 3 x 2 factorial scheme, with 2 soil phosphate levels (40 e 200 mg P dm(-3) soil), combined with 3 nutrient sources applied via foliar sprays (potassium phosphite, potassium phosphate, and potassium chloride as a control), and 2 foliar application numbers (single and two application). Additional treatments showed that phosphite is not P source for common bean nutrition. Phosphite supply in soil increased the P content in shoot (at full physiological maturity stage) and grains, but at the same time considerably decreased grain yield, regardless of the soil phosphate availability. Foliar sprays of phosphite decreased grain yield in plants grown under low soil phosphate availability, but no effect was observed in plants grown under adequate soil phosphate availability. In general, foliar sprays of phosphate did not satisfactorily improve grain yield of the common bean plants grown under low soil phosphate availability.

Segregated anatomical input to sub-regions of the rodent superior colliculus associated with approach and defense

The superior colliculus (SC) is responsible for sensorimotor transformations required to direct gaze toward or a way from unexpected, biologically salient events. Significant changes in the external world are signaled to SC through primary multisensory afferents, spatially organized according to a retinotopic topography. For animals, where anunexpected event could indicate the presence of either predator or prey, early decisions to approach or avoid are particularly important. Rodents' ecology dictates predators are most often detected initially as movements in upper visual field (mapped in medial SC), while appetitive stimuli are normally found in lower visual field (mapped in lateral SC). Our purpose was to exploit this functional segregation to reveal neural sites that can bias or modulate initial approach or avoidance responses. Small injections of Fluoro-Gold were made into medial or lateral sub-regions of intermediate and deep layers of SC (SCm/SCl). A remarkable segregation of input to these two functionally defined areas was found. (i) There were structures that projected only to SCm (e.g., specific cortical areas, lateral geniculate and suprageniculate thalamic nuclei, ventromedial and premammillary hypothalamic nuclei, and several brain-stem areas) or SCl (e.g., primary somatosensory cortex representing upper body parts and vibrissae and parvicellular reticular nucleus in the brainstem). (ii) Other structures projected to both SCm and SCl but from topographically segregated populations of neurons (e.g., zona incerta and substantia nigra pars reticulata). (iii) There were a few brainstem areas in which retrogradely labeled neurons were spatially overlapping (e.g., pedunculopontine nucleus and locus coeruleus). These results indicate significantly more structures across the rat neuraxis are in a position to modulate defense responses evoked from SCm, and that neural mechanisms modulating SC-mediated defense or appetitive behavior are almost entirely segregated.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Strategies in identifying individuals in a segregant population of common bean and implications of genotype x environment interaction in the success of selection

The objective of this study was to compare the BLUP selection method with different selection strategies in F-2:4 and assess the efficiency of this method on the early choice of the best common bean (Phaseolus vulgaris) lines. Fifty-one F-2:4 progenies were produced from a cross between the CVIII8511 x RP-26 lines. A randomized block design was used with 20 replications and one-plant field plots. Character data on plant architecture and grain yield were obtained and then the sum of the standardized variables was estimated for simultaneous selection of both traits. Analysis was carried out by mixed models (BLUP) and the least squares method to compare different selection strategies, like mass selection, stratified mass selection and between and within progeny selection. The progenies selected by BLUP were assessed in advanced generations, always selecting the greatest and smallest sum of the standardized variables. Analyses by the least squares method and BLUP procedure ranked the progenies in the same way. The coincidence of the individuals identified by BLUP and between and within progeny selection was high and of the greatest magnitude when BLUP was compared with mass selection. Although BLUP is the best estimator of genotypic value, its efficiency in the response to long term selection is not different from any of the other methods, because it is also unable to predict the future effect of the progenies x environments interaction. It was inferred that selection success will always depend on the most accurate possible progeny assessment and using alternatives to reduce the progenies x environments interaction effect.

MODELING OF WEEDS INTERFERENCE PERIODS IN BEAN

The research objective was to determine the effects of spacing and seeding density of common bean to the period prior to weed interference (PPI) and weed period prior to economic loss (WEEPPEL). The treatments consisted of periods of coexistence between culture and the weeds, with 0 to 10, 0 to 20, 0 to 30, 0 to 40, 0 to 50, 0 to 60, 0 to 70, and 0 to 80 days and a control maintained without weeds. In addition to the periods of coexistence, there were still studies with an inter-row of 0.45 and 0.60 m, 10 and 15 plants m(-1). The experimental delineation used was randomized blocks with four repetitions per treatment. The grain productivity of the culture had a reduction of 63, 50, 42 and 57% when the coexistence with the weed plants was during the entire cycle of the culture for a row spacing of 0.45 m and a seeding density of 10 and 15 plants per meter; and a row spacing of 0.60m and a seeding density of 10 and 15 plants per meter, respectively. The PPI occurred in 23, 27, 13, and 19 days after crop emergence and WEEPPEL in 10, 9, 8, and 8 days, respectively.

Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat

The mesopontine rostromedial tegmental nucleus (RMTg) is a mostly ?-aminobutyric acid (GABA)ergic structure believed to be a node for signaling aversive events to dopamine (DA) neurons in the ventral tegmental area (VTA). The RMTg receives glutamatergic inputs from the lateral habenula (LHb) and sends substantial GABAergic projections to the VTA, which also receives direct projections from the LHb. To further specify the topography of LHb projections to the RMTg and VTA, small focal injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin were aimed at different subdivisions of the LHb. The subnuclear origin of LHb inputs to the VTA and RMTg was then confirmed by injections of the retrograde tracer cholera toxin subunit b into the VTA or RMTg. Furthermore, we compared the topographic position of retrogradely labeled neurons in the RMTg resulting from VTA injections with that of anterogradely labeled axons emerging from the LHb. As revealed by anterograde and retrograde tracing, LHb projections were organized in a strikingly topographic manner, with inputs to the RMTg mostly arising from the lateral division of the LHb (LHbL), whereas inputs to the VTA mainly emerged from the medial division of the LHb (LHbM). In the RMTg, profusely branched LHb axons were found in close register with VTA projecting neurons and were frequently apposed to the latter. Overall, our findings demonstrate that LHb inputs to the RMTg and VTA arise from different divisions of the LHb and provide direct evidence for a disynaptic pathway that links the LHbL to the VTA via the RMTg. J. Comp. Neurol. 520:12781300, 2012. (C) 2011 Wiley Periodicals, Inc.

Psychiatric comorbidities in temporal lobe epilepsy: Possible relationships between psychotic disorders and involvement of limbic circuits

Objective: Mounting evidence suggests that the limbic system is pathologically involved in cases of psychiatric comorbidities in temporal lobe epilepsy (TLE) patients. Our objective was to develop a conceptual framework describing how neuropathological and connectivity changes might contribute to the development of psychosis and to the potential neurobiological mechanisms that cause schizophrenia-like psychosis in TLE patients. Methods: In this review, clinical and neuropathological findings, especially brain circuitry of the limbic system, were examined together to enhance our understanding of the association between TLE and psychosis. Finally, the importance of animal models in epilepsy and psychiatric disorders was discussed. Conclusions: TLE and psychiatric symptoms coexist more frequently than chance would predict. Damage and deregulation among critical anatomical regions, such as the hippocampus, amygdala, thalamus, and the temporal, frontal and cingulate cortices, might predispose TLE brains to psychosis. Studies of the effects of kindling and injection of neuroactive substances on behavior and electrophysiological patterns may offer a model of how limbic seizures in humans increase the vulnerability of TLE patients to psychiatric symptoms.

Changes in spatial and temporal gene expression during incompatible interaction between common bean and anthracnose pathogen

Common bean, one of the most important legumes for human consumption, may have drastic reduction in yield due to anthracnose, a disease caused by the fungus Colletotrichum lindemuthianum. Rapid induction of the plant defense mechanisms is essential to establish an incompatible interaction with this pathogenic fungus. In this study, we evaluated spatial (leaves, epicotyls and hypocotyls) and temporal (24, 48, 72 and 96 hours after inoculation [HAI]) relative expression (RE) of 12 defense-related transcripts selected from previously developed ESTs libraries, during incompatible interaction between the resistant common bean genotype SEL 1308 and the avirulent anthracnose pathogen race 73, using real time quantitative RT-PCR (RT-qPCR) analysis. All selected transcripts, including the ones coding for pathogenesis-related (PR) proteins (PR1a, PR1b, PR2, and PR16a and PR16b) were differentially regulated upon pathogen inoculation. The expression levels of these transcripts were dependent on the tissue and time post inoculation. This study contributes to a better understanding of the kinetics of induced defenses against a fungal pathogen of common bean and may be used as a base line to study defenses against a broad range of pathogens including bacteria as well as non-host resistance. (C) 2012 Elsevier GmbH. All rights reserved.

The periaqueductal gray as a critical site to mediate reward seeking during predatory hunting

Previous studies using morphine-treated dams reported a role for the rostral lateral periaqueductal gray (rIPAG) in the behavioral switching between nursing and insect hunting, likely to depend on an enhanced seeking response to the presence of an appetitive rewarding cue (i.e., the roach). To elucidate the neural mechanisms mediating such responses, in the present study, we first observed how the rIPAG influences predatory hunting in male rats. Our behavioral observations indicated that bilateral rIPAG NMDA lesions dramatically interfere with prey hunting, leaving the animal without chasing or attacking the prey, but do not seem to affect the general levels of arousal, locomotor activity and regular feeding. Next, using Phaseolus vulgaris-leucoagglutinin (PHA-L), we have reviewed the rIPAG connection pattern, and pointed out a particularly dense projection to the hypothalamic orexinergic cell group. Double labeled PHA-L and orexin sections showed an extensive overlap between PHA-L labeled fibers and orexin cells, revealing that both the medial/perifornical and lateral hypothalamic orexinergic cell groups receive a substantial innervation from the rIPAG. We have further observed that both the medial/perifornical and lateral hypothalamic orexinergic cell groups up-regulate Fos expression during prey hunting, and that rIPAG lesions blunted this Fos increase only in the lateral hypothalamic, but not in the medial/perifornical, orexinergic group, a finding supposedly associated with the lack of motivational drive to actively pursue the prey. Overall, the present results suggest that the rIPAG should exert a critical influence on reward seeking by activating the lateral hypothalamic orexinergic cell group. (C) 2011 Elsevier B.V. All rights reserved.

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.