Leaf Apoplast Research Articles

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast.

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Early changes of the pH of the apoplast are different in leaves, stem and roots of Vicia faba L. under declining water availability

Changes in pH of the apoplast have recently been discussed as an important factor in adjusting transpiration and water relations under conditions of drought via modulatory effect on abscisic acid (ABA) concentration. Using Vicia faba L., we investigated whether changes in the root, shoot and leaf apoplastic pH correlated with (1) a drought-induced reduction in transpiration and with (2) changes in ABA concentration. Transpiration, leaf water potential and ABA in leaves were measured and correlated with root and shoot xylem pH, determined by a pH microelectrode, and pH of leaf apoplast quantified by microscopy-based in vivo ratiometric analysis. Results revealed that a reduction in transpiration rate in the early phase of soil drying could not be linked with changes in the apoplastic pH via effects on the stomata-regulating hormone ABA. Moreover, drought-induced increase in pH of xylem or leaf apoplast was not the remote effect of an acropetal transport of alkaline sap from root, because root xylem acidified during progressive soil drying, whereas the shoot apoplast alkalized. We reason that other, yet unknown signalling mechanism was responsible for reduction of transpiration rate in the early phase of soil drying.

Exogenous Ca2SiO4 enrichment reduces the leaf apoplastic Na+ and increases the growth of okra (Abelmoschus esculentus L.) under salt stress

Salt-sensitive plants have been hypothesized to deposit excessive Na+ in their leaf apoplast, which can mainly disturb shoot growth of crop plants. Knowledge of the subcellular location of Na+ is important to ascertain its toxicity level under salt stress. However, this hypothesis of extracellular salt deposition under salinity stress is still not fully understood. This research mainly focuses on the influence of 1mM calcium silicate (Ca2SiO4) on growth and Na+ accumulation in the leaf apoplast of hydroponically grown salt sensitive okra plants under short-term stress of 100mM NaCl. It has been found that Na+ concentration not only increases in total shoot and root of okra, but also in the leaf apoplast of salt-stressed plants. Na+ concentration was significantly reduced in total shoot (52%) and leaf apoplast (30%), when 1mM Ca2SiO4 was applied as a foliar plus pot under salt stress. Moreover, calcium silicate treated plants showed significant improvement in shoot fresh weight, shoot length, leaf area and leaf length under 100mM NaCl stress. Together, these results suggest that calcium silicate enrichment in pot and as a foliar spray increases the growth of Abelmoschus esculentus by reducing the Na+ concentration in the leaf apoplast, which could thus ameliorate the negative impacts of salt stress on plants.

Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins.

Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses.

English

Agriculture is one of the most exposed sectors to vagaries of climate change (Malik, 2012). Around 20-25% of the world (Munns and Tester, 2008) and 26% of Pakistan’s agricultural (Anonymous, 2010) irrigated lands are affected by salinity. Saline area is increasing day by day due to higher evapotranspiration that demand more irrigation and consequently, more salt accumulation in the root zone especially in arid and semiarid regions of the world (Iqbal et al., 2009; Mou, 2011). Predominately, salinization (50–80%) is caused by NaCl salt (Kaouther, 2012). Primary toxic ion is Na as it not only impairs K uptake but also interrupts regulation of stomata which eventually causes water loss. Na enters in leaf apoplast through xylem stream and left behind as water evaporates. Na mainly compete and occupies cations binding sites by reducing uptake and transport of Ca and K (Munns and Tester, 2008; Horie et al., 2012; Hasanuzzaman et al., 2013). Hyperosmotic accompanied with hyper ionic conditions enhance generation of reactive oxygen species (ROS) damaging the proteins, lipids, DNA and carbohydrates molecules, which weakens the plant defense mechanism and modifies membrane structures and its composition (Tuteja, 2007; Ismail, 2014; Jbir-Koubaa, 2014; Gao et al., 2015). Moreover, ROS intensifies MDA contents, deactivates enzymes, disrupts ions of normal cellular metabolism and enhances electrolyte leakage that causes programmed cell death (necroptosis) and reduced photosynthetic activity (Gao et al., 2015). Therefore, plants manifest various scavenging machineries like enzymatic antioxidant system (SOD, CAT, POD), the level of which is elevated during abiotic stresses (Gill and Tuteja, 2010; Choudhury, 2013; Ismail, 2014). It also affects plant water relations and gas exchange attributes together with metabolic toxicity, reduction in green pigments and thereby intervening photosynthetic activity (Ashraf and Harris, 2013; Gupta and Huang, 2014; Li et al., 2014). Potato (Solanum tuberosum L.) is a staple food for about 50% world’s population and third largest crop in human consumption. It is fourth major crop with respect to area and production in the world. It provides high energy per unit land, water and time along-with valuable source of vitamins and minerals (Abhayapala et al., 2014; Gao et al., 2015). It is moderately salt-sensitive (Mitsuya et al., 2000) with 50% growth and yield reduction at 5 dSm salt stress (Hmida-Sayari et al., 2005). However, tolerance level varies from cultivar to cultivar (Bruns and HechtBuchholz, 1990). Increasing saline area demands salt tolerant cultivars for sustainable potato production (Mou, 2011). Hence, it is necessary to understand salinity tolerance mechanism in potato, helpful in developing stress tolerant potato cultivars by using various modern techniques (Gururani et al., 2013). Considering this scenario, present experiment was conducted to assess adverse effects of salinity stress on ionic Pak. J. Agri. Sci., Vol. 53(1), 17-25; 2016 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI: 10.21162/PAKJAS/16.4766 http://www.pakjas.com.pk

Collection of Apoplastic Fluids from Arabidopsis thaliana Leaves.

The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite content in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described.

English

The purpose of this experiment was to relate the tolerance of Commelina benghalensis to two different kinds of glyphosate formulation, in function of the absorption time and translocation of this molecule in the cells of this plant. The experiment was conducted in a greenhouse. The experimental design is a completely randomized design (CRD), with four treatments follows; glyphosate potassium salt and isopropylamine tested with commercial dosage, haematoxylin dye and a control, with three repetitions. Sample from the leaves, stems and roots were collected and subdivided in twenty-five subsamples. The evaluations were performed by microscopic views every 30 min, until the herbicide was absorbed into the parenchyma of the mesophyll. After 30 min of the application of isopropylamine glyphosate, there was accumulation of this molecule inside leaf apoplast which indicated the process of penetration of isopropylamine glyphosate. For the glyphosate potassium salt the absorption was observed 2 h after application. After 7 h, the presence of a small concentration of the product in the stem was verified. 12 h later, a low concentration was verified in the root. After 36 h the phloem was full of the isopropyl amine salt glyphosate, 86 h later its presence in the xylem and phloem, together with the symptoms of destruction of the parenchyma tissue was visualized. The solution with isopropyl amine salt, after 7 h, was seen in the phloem, with a 12 h concentration of isopropyl amine salt decrease in the stem, which overloaded the phloem of the root. After 86 h, all the parenchyma tissue of the xylem and phloem were destructed. The absorption and translocation of glyphosate depended on its formulation, which represented more or less tolerance of the plant to the herbicide, but tolerance was not the only factor influenced by anatomy, the physiological, biochemical and genetic features were also critical.   Key words: Phloem, morphology, tropical spiderwort.

A Large Repetitive RTX-Like Protein Mediates Water-Soaked Lesion Development, Leakage of Plant Cell Content and Host Colonization in the Pantoea stewartii subsp. stewartii Pathosystem.

Pantoea stewartii subsp. stewartii is the etiological agent of Stewart's wilt and is a serious bacterial pathogen affecting sweet corn. During the leaf blight phase, P. stewartii colonizes the leaf apoplast and causes a characteristic water-soaked lesion. The Hrp type III secretion system has been implicated in the water-soaking phenotype, and the goal of this study was to investigate other potential factors that contribute to the plant cellular disruption associated with these lesions. The P. stewartii genome contains a gene encoding a large repetitive RTX toxin, designated rtx2. RTX toxins comprise a large family of pore-forming proteins, which are widely distributed among gram-negative bacteria. These cytotoxins usually lyse their target host cells and cause significant tissue damage as a consequence. We hypothesized that this RTX-like toxin plays a role in the water-soaking phase of infection due to its predicted cytolytic properties. Based on the data reported here, we conclude that RTX2 contributes significantly to the development of water-soaked lesions and leakage of plant cellular contents and is an important pathogenicity factor for P. stewartii.

High atmospheric carbon dioxide-dependent alleviation of salt stress is linked to RESPIRATORY BURST OXIDASE 1 (RBOH1)-dependent H2O2 production in tomato (Solanum lycopersicum).

Plants acclimate rapidly to stressful environmental conditions. Increasing atmospheric CO2 levels are predicted to influence tolerance to stresses such as soil salinity but the mechanisms are poorly understood. To resolve this issue, tomato (Solanum lycopersicum) plants were grown under ambient (380 μmol mol(-1)) or high (760 μmol mol(-1)) CO2 in the absence or presence of sodium chloride (100mM). The higher atmospheric CO2 level induced the expression of RESPIRATORY BURST OXIDASE 1 (SlRBOH1) and enhanced H2O2 accumulation in the vascular cells of roots, stems, leaf petioles, and the leaf apoplast. Plants grown with higher CO2 levels showed improved salt tolerance, together with decreased leaf transpiration rates and lower sodium concentrations in the xylem sap, vascular tissues, and leaves. Silencing SlRBOH1 abolished high CO2 -induced salt tolerance and increased leaf transpiration rates, as well as enhancing Na(+) accumulation in the plants. The higher atmospheric CO2 level increased the abundance of a subset of transcripts involved in Na(+) homeostasis in the controls but not in the SlRBOH1-silenced plants. It is concluded that high atmospheric CO2 concentrations increase salt stress tolerance in an apoplastic H2O2 dependent manner, by suppressing transpiration and hence Na(+) delivery from the roots to the shoots, leading to decreased leaf Na(+) accumulation.

Manganese phytotoxicity: new light on an old problem.

Manganese (Mn) is an essential micronutrient that is phytotoxic under certain edaphic and climatic conditions. Multiple edaphic factors regulate Mn redox status and therefore its phytoavailability, and multiple environmental factors including light intensity and temperature interact with Mn phytotoxicity. The complexity of these interactions coupled with substantial genetic variation in Mn tolerance have hampered the recognition of Mn toxicity as an important stress in many natural and agricultural systems. Conflicting theories have been advanced regarding the mechanism of Mn phytotoxicity and tolerance. One line of evidence suggests that Mn toxicity ocurrs in the leaf apoplast, while another suggests that toxicity occurs by disruption of photosynthetic electron flow in chloroplasts. These conflicting results may at least in part be attributed to the light regimes employed, with studies conducted under light intensities approximating natural sunlight showing evidence of photo-oxidative stress as a mechanism of toxicity. Excessive Mn competes with the transport and metabolism of other cationic metals, causing a range of induced nutrient deficiencies. Compartmentation, exclusion and detoxification mechanisms may all be involved in tolerance to excess Mn. The strong effects of light, temperature, precipitation and other climate variables on Mn phytoavailability and phytotoxicity suggest that global climate change is likely to exacerbate Mn toxicity in the future, which has largely escaped scientific attention. Given that Mn is terrestrially ubiquitous, it is imperative that the heightened risk of Mn toxicity to both managed and natural plant ecosystems be factored into evaluation of the potential impacts of global climate change on vegetation. Large inter- and intraspecific genetic variation in tolerance to Mn toxicity suggests that increased Mn toxicity in natural ecosystems may drive changes in community composition, but that in agroecosystems crops may be developed with greater Mn tolerance. These topics deserve greater research attention.

Chloride-inducible transient apoplastic alkalinizations induce stomata closure by controlling abscisic acid distribution between leaf apoplast and guard cells in salt-stressed Vicia faba.

Chloride stress causes the leaf apoplast transiently to alkalize, an event that is presumed to contribute to the ability of plants to adapt to saline conditions. However, the initiation of coordinated processes downstream of the alkalinization is unknown. We hypothesize that chloride-inducible pH dynamics are a key chemical feature modulating the compartmental distribution of abscisic acid (ABA) and, as a consequence, affecting stomata aperture. Apoplastic pH and stomata aperture dynamics in intact Vicia faba leaves were monitored by microscopy-based ratio imaging and porometric measurements of stomatal conductance. ABA concentrations in leaf apoplast and guard cells were compared with pH dynamics by gas-chromatography-mass-spectrometry (GC-MS) and liquid-chromatography-tandem-mass spectrometry (LC-MS/MS). Results demonstrate that, upon chloride addition to roots, an alkalizing factor that initiates the pH dynamic propagates from root to leaf in a way similar to xylem-distributed water. In leaves, it induces a systemic transient apoplastic alkalinization that causes apoplastic ABA concentration to increase, followed by an elevation of endogenous guard cell ABA. We conclude that the transient alkalinization, which is a remote effect of chloride stress, modulates the compartmental distribution of ABA between the leaf apoplast and the guard cells and, in this way, is instrumental in inducing stomata closure during the beginning of salinity.

Biosurfactants Have the Potential to Induce Defence Against Phytophthora infestans in Potato

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases worldwide. Currently, its management mainly relies on the frequent use of synthetic chemicals, and there is a need to develop more sustainable strategies. Biosurfactants produced by fluorescent pseudomonads have been shown to rapidly, within 1 min, cause zoospore lyses. This study investigated if the biosurfactant-producing strain Pseudomonas koreensis 2.74 and its biosurfactants have the potential to induce resistance in potato to late blight. The experiments were performed with treatments of whole plants of the susceptible potato cultivar Bintje and the partially resistant cultivar Ovatio. A significant disease reduction and an induced secretion of proteins such as pathogenesis-related (PR) protein 1, within the leaf apoplast, were observed in the potato cultivar Ovatio after treatment with the biosurfactant at a concentration of 1 mg ml �1 . No significant effects on disease development were observed after treatment with the bacterial suspension of Pseudomonas koreensis 2.74.

Urea retranslocation from senescing Arabidopsis leaves is promoted by DUR3-mediated urea retrieval from leaf apoplast.

In plants, urea derives either from root uptake or protein degradation. Although large quantities of urea are released during senescence, urea is mainly seen as a short-lived nitrogen (N) catabolite serving urease-mediated hydrolysis to ammonium. Here, we investigated the roles of DUR3 and of urea in N remobilization. During natural leaf senescence urea concentrations and DUR3 transcript levels showed a parallel increase with senescence markers like ORE1 in a plant age- and leaf age-dependent manner. Deletion of DUR3 decreased urea accumulation in leaves, whereas the fraction of urea lost to the leaf apoplast was enhanced. Under natural and N deficiency-induced senescence DUR3 promoter activity was highest in the vasculature, but was also found in surrounding bundle sheath and mesophyll cells. An analysis of petiole exudates from wild-type leaves revealed that N from urea accounted for >13% of amino acid N. Urea export from senescent leaves further increased in ureG-2 deletion mutants lacking urease activity. In the dur3 ureG double insertion line the absence of DUR3 reduced urea export from leaf petioles. These results indicate that urea can serve as an early metabolic marker for leaf senescence, and that DUR3-mediated urea retrieval contributes to the retranslocation of N from urea during leaf senescence.

Assays to Assess Virulence of Xanthomonas axonopodis pv. manihotis on Cassava

Cassava (Manihot esculenta) is a root crop that provides calories for people living in more than 100 tropical and subtropical countries and serves as a raw material for processing into starch and biofuels as well as feed for livestock (Howeler et al., 2013). Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight (CBB), can cause extensive crop damage (reviewed in Lopez et al., 2012; Lozano, 1986). Bacterial movement, growth in planta and the ability to cause disease symptoms are all important measures of bacterial fitness and plant susceptibility to CBB. Here we present a protocol for visualizing the movement of Xam within the plant. We also provide a detailed method of assaying bacterial growth in the cassava leaf midvein, and bacterial growth and disease symptom development in the leaf apoplast. These methods will be important tools for determining Xam strain pathogenicity and for developing cassava varieties that are resistant to CBB.

Label-free nanoUPLC-MSE based quantification of antimicrobial peptides from the leaf apoplast of Nicotiana attenuata.

BackgroundOverexpressing novel antimicrobial peptides (AMPs) in plants is a promising approach for crop disease resistance engineering. However, the in planta stability and subcellular localization of each AMP should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pI ranges of AMPs which limits the utility of standard proteomic gel-based methods. Despite recent advances in quantitative shotgun proteomics, its potential for AMP analysis has not been utilized and high throughput methods are still lacking.ResultsWe created transgenic Nicotiana attenuata plants that independently express 10 different AMPs under a constitutive 35S promoter and compared the extracellular accumulation of each AMP using a universal and versatile protein quantification method. We coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanoUPLC-MSE analysis using Hi3 method and identified/quantified 7 of 10 expressed AMPs in the transgenic plants ranging from 37 to 91 amino acids in length. The quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of 91 to 254 pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in N. attenuata. The chosen mass spectrometric approach proved to be highly sensitive in the detection of different AMP types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation.ConclusionsHeterologous expression of AMPs in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. The method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0398-9) contains supplementary material, which is available to authorized users.

Extreme climatic events: impacts of drought and high temperature on physiological processes in agronomically important plants

Climate models predict more frequent and more severe extreme events (e.g. heat waves, extended drought periods, flooding) in many regions for the next decades. The impact of adverse environmental conditions on crop plants is ecologically and economically relevant. This review is focused on drought and heat effects on physiological status and productivity of agronomically important plants. Stomatal opening represents an important regulatory mechanism during drought and heat stress since it influences simultaneously water loss via transpiration and CO2 diffusion into the leaf apoplast which further is utilized in photosynthesis. Along with the reversible short-term control of stomatal opening, stomata and leaf epidermis may produce waxy deposits and irreversibly down-regulate the stomatal conductance and non-stomatal transpiration. As a consequence photosynthesis will be negatively affected. Rubisco activase - a key enzyme in keeping the Calvin cycle functional – is heat-sensitive and may become a limiting factor at elevated temperature. The accumulated reactive oxygen species during stress represent an additional challenge under unfavorable conditions. Drought and heat cause accumulation of free amino acids which are partially converted into compatible solutes such as proline. This is accompanied by lower rates of both nitrate reduction and de novo amino acid biosynthesis. Protective proteins (e.g. dehydrins, chaperones, antioxidant enzymes or the key enzyme for proline biosynthesis) play an important role in leaves and may be present at higher levels under water deprivation or high temperatures. On the whole plant level, effects on long-distance translocation of solutes via xylem and phloem and on leaf senescence (e.g. anticipated, accelerated or delayed senescence) are important. The factors mentioned above are relevant for the overall performance of crops under drought and heat and must be considered for genotype selection and breeding programs.

Transcriptional analysis of the global regulatory networks active in Pseudomonas syringae during leaf colonization.

ABSTRACTThe plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean (Phaseolus vulgaris). To understand the contribution of distinct regulators to B728a fitness and pathogenicity, we performed a transcriptome analysis of strain B728a and nine regulatory mutants recovered from the surfaces and interior of leaves and exposed to environmental stresses in culture. The quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator, SalA, formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals and a plant signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. SalA functioned as a central regulator of iron status based on its reciprocal regulation of pyoverdine and achromobactin genes and also sulfur uptake, suggesting a role in the iron-sulfur balance. RetS functioned almost exclusively to repress secondary metabolite genes when the cells were not on leaves. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN, and AlgU in global regulation in B728a in planta and a high level of plasticity in these regulators’ responses to distinct environmental signals.

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: insight into proteins at intracellular and extracellular spaces.

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.