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Root-knot nematodes, genus Meloidogyne, are among the most plant damaging pathogens worldwide. The action of natural products against plant pathogens has been investigated to assess their effectiveness in the control of diseases. Thus, the present study aimed to evaluate the phytochemistry potential of the Ficus species for the control of Meloidogyne javanica. In vitro inhibitory activity assays were performed with crude ethanolic extracts of leaves and branches from 10 Ficus species. Among these, Ficus carica extracts exhibited strong paralysis activity against second stage juveniles (J2) (EC50 = 134.90 μg ∙ ml–1), after 72 hours. In addition, high efficacy was observed in egg-hatching inhibition at different embryonic stages. Microscopy analysis revealed severe morphological alterations in the nematode tissues at the J2 stage, as well as immotility of juveniles released from eggs in the presence of F. carica extracts. The efficacy of the treatments for the other species was very low. These differences were supported by the variation in the compound classes, mainly for alkaloids and metabolite profiles by Gas Chromatography/Mass Spectrometry (GC/MS) when F. carica was compared with the other species. The results indicated that F. carica is a promising source for the isolation and identification of molecules capable of acting in the control of M. javanica.

Embryo development in eggs of the spittlebug Mahanarva spectabilis (Distant) (Hemiptera: Cercopidae) passes through four phases (known as S1 to S4) being stopped at S2 during diapause. Studies about the molecular basis of diapause in spittlebugs are nonexistent. Here, we analyzed proteins from non-diapausing (ND), diapausing (D) and post-diapausing (PD) eggs of the spittlebug M. spectabilis. In total, we identified 87 proteins where 12 were in common among the developmental and diapause phases and 19 remained as uncharacterized. Non-diapausing eggs (S2ND and S4ND) showed more proteins involved in information storage and processing than the diapausing ones (S2D). Eggs in post-diapausing (S4PD) had a higher number of proteins associated with metabolism than S2D. The network of protein interactions and metabolic processes allowed the identification of different sets of molecular interactions for each developmental and diapause phases. Two heat shock proteins (Hsp65 and Hsp70) along with two proteins associated with intracellular signaling (MAP4K and a serine/threonine-protein phosphatase) were found only in diapausing and/or post-diapausing eggs and are interesting targets to be explored in future experiments. These results shine a light on one key biological process for spittlebug survival and represent the first search for proteins linked to diapause in this important group of insects.

Drought stress is major abiotic stress that affects soybean production. Therefore, it is widely desirable that soybean becomes more tolerant to stress. To provide insights into regulatory mechanisms of the stress response, we compared the global gene expression profiles from leaves of two soybean genotypes that display different responses to water-deficit (BR 16 and Embrapa 48, drought-sensitive and drought-tolerant, respectively). After the RNA-seq analysis, a total of 5335 down-regulated and 3170 up-regulated genes were identified in the BR16. On the other hand, the number of genes differentially expressed was markedly lower in the Embrapa 48, 355 up-regulated and 471 down-regulated genes. However, induction and expression of protein kinases and transcription factors indicated signaling cascades involved in the drought tolerance. Overall, the results suggest that the metabolism of pectin is differently modulated in response to drought stress and may play a role in the soybean defense mechanism against drought. This occurs via an increase of the cell wall plasticity and crosslink, which contributed to a higher hydraulic conductance (K(f)) and relative water content (RWC%). The drought-tolerance mechanism of the Embrapa 48 genotype involves remodeling of the cell wall and increase of the hydraulic conductance to the maintenance of cell turgor and metabolic processes, resulting in the highest leaf RWC, photosynthetic rate (A), transpiration (E) and carboxylation (A/C(i)). Thus, we concluded that the cell wall adjustment under drought is important for a more efficient water use which promoted a more active photosynthetic metabolism, maintaining higher plant growth under drought stress. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-021-00043-4.

Despite being described by the nineteenth century naturalist Auguste de Saint-Hilaire as an important native Brazilian medicinal species, Strychnos pseudoquina A. St.-Hil. remains poorly characterized. To preserve biodiversity in the Cerrado hotspot and tackle the negative consequences of S. pseudoquina overharvesting, the possibility of in vitro propagation and callus induction of this species was investigated. Thus, this study has established a novel protocol for: (i) in vitro cultivation of S. pseudoquina using zygotic embryo culture; (ii) callogenesis; and (iii) in vitro plantlet development in three different culture media: Murashige and Skooog (MS), Woody Plant Medium (WPM), and “Juan, Antonio, Diva and Silvia” medium (JADS). This was complemented by a liquid chromatography coupled to mass spectrometry (LC–MS/MS) analysis for the identification of flavonoid profile of ex vitro extracts from S. pseudoquina bark and leaves, as well as extracts from leaves, stems, roots, and root-derived calli of in vitro grown plants to assess the production of secondary metabolites. Results support the feasibility of S. pseudoquina preservation via embryo culturing and subsequent plantlet recovery and development. The use of zygotic embryos reduces substantially the time to generate seedlings. Cultured zygotic embryos germination occurred within 7 days, overcoming S. pseudoquina seed dormancy that usually takes 65 days and, under optimal conditions enables the emergence of true leaves. Additionally, root-derived callus of S. pseudoquina could represent an alternative source for flavonoid production and in vitro conservation of the species. Plant tissue culture techniques can be efficient for the micropropagation and flavonoids production in Strychnos pseudoquina A. St.-Hil.

Soybean plants are exposed to multiple stresses during development. Co-occurring stresses can activate common defense pathways on plants, increasing end products concentrations, such as protease inhibitors (PIs). Here, we evaluated the impact of drought stress on soybean defense mechanisms regarding PIs. Furthermore, the midgut proteolytic activity and survival of A. gemmatalis larvae fed with drought-stressed and well-watered plants were assessed. The lipoxygenase activity remained similar on soybean exposed to moderate (−0.6 and − 1.0 MPa) and severe (−1.6 MPa) drought stresses, however were higher than the irrigated control. The PIs activity on soybean was only induced due to herbivory at those exposed to moderate stress (−0.6 and − 1.0 MPa). The total-proteases and trypsin-like reduced activities in the midgut of A. gemmatalis that fed on moderate-stressed plants reflected the effects of the PIs induced after herbivory. Furthermore, A. gemmatalis showed reduced survival on drought-stressed plants over those well-watered. The results add knowledge about factors that limit A. gemmatalis performance.

Signal perception in plants is determinant to trigger specific physiological changes that confer resistance during the early stages of the insect attack. Thus, molecular characterization of both insect effectors and plant hormonal regulators are essential in developing forage grasses resistant to infestation by spittlebug Mahanarva spectabilis. We evaluated the presence of effectors in the salivary glands of M. spectabilis that could enable the infestation of elephant grass (Pennisetum purpureum). The presence of an NTPDase-like enzyme was confirmed, and higher activity was observed in females that hydrolyzed ADP. High levels of long chain fatty acids, such as octadecanoid acid (19.5%), prostaglandin A2 (4.7%), eicosanoid acid and the phytohormone salicylic acid (~4.0 μg/g) were also detected in the salivary glands. After 24 h of infestation, the phytohormone profiles were altered in leaves damaged by M. spectabilis, suggesting that the insect effectors modulate the plant response. Our results suggest that the plant response modulation may be the result of crosstalk between the salicylic acid, jasmonic acid, abscisic acid and zeatin pathways, induced by molecules from the salivary glands. This shows that the spittlebugs can interfere with jasmonic acid and zeatin accumulation in elephant grass plants.

Insect pests such as Anticarsia gemmatalis cause defoliation and yield losses. Soybean breeding has obtained resistant genotypes, however the mechanism remains unknown. Studies indicated the presence of deterrents compounds in the resistant genotype IAC17, and their leaf metabolite profiles were compared to the susceptible genotype UFV105, which was elicited or not by caterpillar infestation. Cluster analysis indicated a significative distinction between these profiles as well as differences in plant defense pathways. Methylquercetins were constitutively present in the largest concentrations, specifically in the IAC17. Relationship between the resistance and the levels of phytohormones jasmonic acid, abscisic acid and salicylic acid was not observed. However, 1-aminocyclopropane -1carboxylic acid levels indicated that the ethylene may be involved in the constitutive biosynthesis of bioactive compounds. Extracts were added to the diets at three different concentrations to evaluate the effect on caterpillar survival. Lowest survival rates were observed when extracts from the resistant IAC 17 were used, at the lowest concentrations. Survival rates were not higher when IAC 17 infested by caterpillars were used. On the other hand, when extracts from the susceptible were used, the survival reductions were only observed in the highest extract concentrations. These supplementations of the diet reduced the digestive capacity, agreeing with the proteolytic activities, whereas malformations of the intestinal cells were dose dependent. The inhibitory effects persisted in higher dilutions only for the IAC17. Constitutive resistance was also explained by higher levels of protease inhibition. These results can be useful to elucidate the genes and cascades controlling the resistance.

Chemical pesticides are the main tool used to handle lepidopteran insects in agriculture. However, in addition to causing environmental problems, the insects have acquired resistance to these molecules. Thus, the development of environmentally friendly biopesticides with higher specificity, such as organic protease inhibitors, has been encouraged. This work evaluated the inhibitory effects of two tripeptides, Pep1 and Pep2, obtained by removing the cleavage site of tripeptidyl substrates for trypsin-like proteases. Anticarsia gemmatalis were used as biochemical model and the trypsin-like activity of larvae exposed to different dosages of the tripeptides and protease inhibitors merged in an artificial diet was evaluated. Further, we selected one dose of each tripeptide to set a biological assay, where the total-proteolytic, cysteine-proteases, chymotrypsin-like and trypsin-like activities were determined. The tripeptides shown to have potential to inhibit trypsin-like serine proteases of A. gemmatalis midgut, being the lowest activity detected at doses of 170.4 μM and 251.2 μM for Pep1 and Pep2, respectively. The survival curves obtained using Kaplan-Meier estimators indicated that Pep2 and Pep1 affected survival when comparing to the control group. However, the effect of Pep1 on survival was more pronounced indicating the lowest percent of survival at the end of the larval phase (20%). In addition, larvae exposed to Pep1 maintained normal total proteolytic activity at the expense of the activities of chymotrypsin-like and cysteine proteases. Collectively, our findings revealed that the tripeptides, especially Pep1, exhibit toxic effects on A. gemmatalis and should be tested against other lepidopteran insects.

Animal venom peptides have been reported as potential antimicrobial molecules for the development of novel antibiotics against resistant bacteria. Recent studies indicate that ant venoms is an important source of novel antimicrobial peptides. In the present study, the antimicrobial activity of the venom of the predatory ant Pachycondyla striata Smith, 1858, was evaluated against some pathogenic bacteria of medical importance. Growth inhibition tests revealed that the venom has broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. Mass spectrometry analysis of the fractions with antimicrobial activity allowed the identification of venom allergen and venom allergen 5 as well as a peptide of unknown sequence. These three compounds exhibited antibacterial activity against the tested human pathogens.

The symbiosis relationship between arthropods and microorganisms is common in nature and may aid herbivores to counteract plant defences and increase fitness. The spider mite Tetranychus evansi is ...

The spittlebugs Mahanarva spectabilis economically challenges cattle production of neotropical regions, due to its voracious feeding on tropical grasses. Here, we evaluated biochemical responses of the interaction between M. spectabilis and the widely cultivated tropical grasses Brachiaria spp. (i.e., brizantha and decumbens) and elephant grasses (cvs. Roxo de Botucatu and Pioneiro), regarding lipoxygenases, protease inhibitors, phytohormones, and proteolytic activities in the midgut of M. spectabilis. The M. spectabilis-infested grasses increased lipoxygenases activity, except for cv. Pioneiro. The levels of the phytohormones jasmonic and abscisic acids were similarly low in all genotypes and increased under herbivory. Furthermore, salicylic acid concentration was constitutively higher in Brachiaria sp., increasing only in spittlebug-infested B. decumbens. M. spectabilis infestations did not induce increases of protease inhibitors in any forage grass type. The trypsin activity remained unaltered, and the total proteolytic activity increased only in B decumbens-fed insects. Our findings revealed that most forage grasses exposed to spittlebugs activate the lipoxygenases pathway, resulting in increased abscisic and jasmonic acids. However, greater amounts of these hormones do not induce protease inhibitory activity in response to spittlebug attack. This knowledge certainly helps to guide future projects aiming at reducing the impact of spittlebugs on forage production. Made available in DSpace on 2021-09-15T12:00:27Z (GMT). No. of bitstreams: 1 Differential-defense.pdf: 966114 bytes, checksum: 1aff97283378e2d95870009591ee6794 (MD5) Previous issue date: 2021

Metabolomics provide a qualitative and quantitative overview of the metabolites present in organisms, organs, tissues or specific cells. Different metabolomics approaches have been used for phenotyping populations and, together with mapping methods, allowed the identification of DNA sections associated with metabolite levels, defined as metabolic quantitative trait loci (mQTL). Many studies have provided a large number of mQTL and often candidate genes related to distinct metabolic traits. Since plant growth and development as well as responses to environmental changes lead to metabolic adjustments, the understanding of the genetic bases for metabolite changes is fundamental for plant physiology studies being also highly useful for plant breeding programs. Here, we revisited the progress on metabolite analysis currently used to characterize plant metabolite profile. We further describe few examples of the integration between metabolomic approaches and mapping methods allowing mQTL identification and its importance for plant physiology.

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.

Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

Background: Herbivores cause biochemical, physiological, and morphological changes on plants. The ultimate effects of the herbivore activities are damages on the plant's body that can lead from local tissues death to the plant death. The intensity of herbivore attack and the plant's capability to defend itself determine the outcomes of herbivory. When the plant under attack has economic importance, such damages can diminish the productivity of the plantation and compromise the economy of families and the local communities. Although the adverse effects of herbivory on plants and economy are entirely known, some biological mechanisms underlying these damages remain unclear, especially at the anatomical and hormonal levels. The psyllid Triozoida limbata (Hemiptera: Triozidae) is a critical guava (Psidium guajava) pest in the Neotropical region, causing productivity plantation losses up to 55%. Psyllids cause visible morphological changes on guava leaves; however, there is a lack of detailed studied of the cellular, anatomical, and hormonal aspects of the subject. Thus, this work aimed to determine the alterations caused in guava leaves by T. limbata through anatomical and hormonal analyzes. Results: We found that the psyllid's nymphs feeding causes cells hypertrophy and hyperplasia determining the formation of the leaf-roll galls. Although there was no anatomical differentiation of the mesophilic tissues, the cells accumulate starch. Along with the anatomical changes, we observed increasing in the jasmonic acid and reduction of zeatin concentrations in the leaves. Conclusions: The alteration of phytohormone contents and the anatomical changes indicates that the feeding of T. limbata activates plant defenses, but it is not active toward the insects off. The insect nymphs live inside the leaf-roll galls until adults' phase, feeding on the guava leaves xylem and phloem. In commercial guava orchard, the control of T. limbata nymphs have been difficult. Thus, the understanding of the composition of this protective layer can help to find the most effective and efficient control solution for this pest.

International audience; We tested a new approach to describe how a sublethal dose of the Euphorbia milii Des Moul. latex can affect cercariae, inducing changes in the adult Schistosoma mansoni Sambon 1907. We analyzed the effects on biology and used proteomic tools to identify the expression of different proteins. The treatment reduced the development of adults (males, 74.1%; females, 76.3%) and decreased the number of eggs released in 89.9%. The protein profile between adult males of S. mansoni from the control and exposed groups detected 1,020 spots, 26 were differently expressed and 10 were identified and validated. The proteins downregulated after exposition included actin, 14-3-3 epsilon and aldehyde dehydrogenase. The hsp70 was the only protein that was upregulated. The changes in the proteomic profile of adult males of S. mansoni may be related to a muscular fragility of the body. This work provides new insight for schistosomiasis control using a natural, water-soluble product at sublethal concentrations for mollusks. This approach is inexpensive, ecological and efficient as an indirect therapy for schistosomiasis.; Hemos probado un nuevo enfoque para describir cómo una dosis subletal del látex de Euphorbia milii Des Moul. puede afectar a las cercarias, induciendo cambios en el Schistosoma mansoni Sambon 1907 adulto. Se analizaron los efectos sobre la biología y se utilizaron herramientas proteómicas para identificar la expresión de diferentes proteínas. El tratamiento redujo el desarrollo de adultos (machos 74,1%, hembras 76,3%) y disminuyó el número de huevos liberados en 89,9%. El perfil proteico entre machos adultos de S. mansoni de los grupos control y expuestos detectó 1.020 spots, 26 se expresaron de forma diferente y 10 fueron identificados y validados. Las proteínas menos abundante después de la exposición incluyeron actina, 14-3-3 epsilon y aldehído deshidrogenasa. La hsp70 fue la única proteína que se más abundante. Los cambios en el perfil proteómico de los machos adultos de S. mansoni pueden estar relacionados con una fragilidad muscular del cuerpo. Este trabajo proporciona una nueva visión para el control de la esquistosomiasis utilizando un producto natural, soluble en agua a concentraciones subletales para moluscos. Este enfoque es barato, ecológico y eficiente como terapia indirecta para la esquistosomiasis

Drought is one of the major constraints for soybean production in Brazil. In this study we investigated the physiological traits of two soybean parental genotypes under progressive soil drying and rewetting. The plants were evaluated under full irrigation (control) conditions and under water deficit imposed by suspending irrigation until the plants reached predawn leaf water potentials (Ψam) of −1.0 MPa (moderate) and −1.5 MPa (severe). Physiological analyses showed that these genotypes exhibit different responses to water deficit. The Embrapa 48 genotype reached moderate and severe water potential two days after the BR16 genotype and was able to maintain higher levels of A, ETR and ΦPSII even under deficit conditions. This result was not related to changes in gs, 13C isotopic composition and presence of a more efficient antioxidant system. In addition, Fv/Fm values did not decrease in Embrapa 48 genotype in relation to irrigated condition showing that stress was not causing photochemical inhibition of photosynthesis. The greater reduction in the relative growth of the shoots, with concomitant greater growth of the root system under drought, indicates that the tolerant genotype is able to preferentially allocated carbon to the roots, presenting less damage to photosynthesis. Therefore, the physiological responses revealed that the tolerant genotype postponed leaf dehydration by a mechanism involving a more efficient use and translocation of water from root to shoot to maintain cell homeostasis and photosynthetic metabolism under stress.

Climate changes are promoting increases in global temperatures, variability in precipitation, and more frequent insect pest outbreaks. Understanding the molecular mechanisms for the tolerance of both, biotic and abiotic stresses, is critical for the development of improved genotypes and sustainability of productivity under climate change. Thus, we examine the regulatory cascades and metabolic pathways triggered by both drought and insect attack signals. Profiles from the genotypes drought-tolerant (EMBRAPA 48) and resistant to A. gemmatalis infestation (IAC17) were compared with one sensitive to drought/insect attack (BR16). EMBRAPA 48 and IAC17 plants under drought were less susceptible to insect attack, promoting lower caterpillar survival. Survival reductions were not dependent on the drought-tolerance or insect-resistance phenotypes, despite being more pronounced for IAC17. Furthermore, metabolites profiles, gene expression and enzymatic assays lead us to conclude that drought signal by itself was not enough to explain the survival reductions. Protease inhibition activities and expressions of LOX and PI genes correlated with ABA levels, indicating that JA-signalling was potentialized by ABA to enhance the production of deterrent metabolites. Thus, increased ABA levels during drought treatment may be acting synergistically to induce cascades responsive to JA. Thus, regulatory molecular hub(s) integrating signals may be a target to the genetic engineering of plants with multiple tolerances to environmental stresses.

Biotic and abiotic environmental stresses have limited the increase in soybean productivity. Overexpression of the molecular chaperone BiP in transgenic plants has been associated with the response to osmotic stress and drought tolerance by maintaining cellular homeostasis and delaying hypersensitive cell death. Here, we evaluated the metabolic changes in response to the hypersensitivity response (HR) caused by the non-compatible bacteria Pseudomonas syringae pv. tomato in BiP-overexpressing plants. The HR-modified metabolic profiles in BiP-overexpressing plants were significantly distinct from the wild-type untransformed. The transgenic plants displayed a lower abundance of HR-responsive metabolites as amino acids, sugars, carboxylic acids and signal molecules, including p-aminobenzoic acid (PABA) and dihydrosphingosine (DHS), when compared to infected wild-type plants. In contrast, salicylic acid (SA) biosynthetic and signaling pathways were more stimulated in transgenic plants, and both pathogenesis-related genes (PRs) and transcriptional factors controlling the SA pathway were more induced in the BiP-overexpressing lines. Furthermore, the long-chain bases (LCBs) and ceramide biosynthetic pathways showed alterations in gene expression and metabolite abundance. Thus, as a protective pathway against pathogens, HR regulation by sphingolipids and SA may account at least in part by the enhanced resistance of transgenic plants. GmNAC32 transcriptional factor was more induced in the transgenic plants and it has also been reported to regulate flavonoid synthesis in response to SA. In fact, the BiP-overexpressing plants showed an increase in flavonoids, mainly prenylated isoflavones, as precursors for phytoalexins. Our results indicate that the BiP-mediated acceleration in the hypersensitive response may be a target for metabolic engineering of plant resistance against pathogens.

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Drought is one of major constraints that limits agricultural productivity. Some factors, including climate changes and acreage expansion, indicates towards the need for developing drought tolerant genotypes. In addition to its protective role against endoplasmic reticulum (ER) stress, we have previously shown that the molecular chaperone binding protein (BiP) is involved in the response to osmotic stress and promotes drought tolerance. Here, we analyzed the proteomic and metabolic profiles of BiP-overexpressing transgenic soybean plants and the corresponding untransformed line under drought conditions by 2DE-MS and GC/MS. The transgenic plant showed lower levels of the abscisic acid and jasmonic acid as compared to untransformed plants both in irrigated and non-irrigated conditions. In contrast, the level of salicylic acid was higher in transgenic lines than in untransformed line, which was consistent with the antagonistic responses mediated by these phytohormones. The transgenic plants displayed a higher abundance of photosynthesis-related proteins, which gave credence to the hypothesis that these transgenic plants could survive under drought conditions due to their genetic modification and altered physiology. The proteins involved in pathways related to respiration, glycolysis and oxidative stress were not signifcantly changed in transgenic plants as compared to untransformed genotype, which indicate a lower metabolic perturbation under drought of the engineered genotype. The transgenic plants may have adopted a mechanism of drought tolerance by accumulating osmotically active solutes in the cell. As evidenced by the metabolic profiles, the accumulation of nine primary amino acids by protein degradation maintained the cellular turgor in the transgenic genotype under drought conditions. Thus, this mechanism of protection may cause the physiological activities including photosynthesis to be active under drought conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-019-00643-x) contains supplementary material, which is available to authorized users.

Drought is the major abiotic stress which contributes to the reduction in the productivity of soybeans in Brazil, especially in the savanna regions. Two parental genotypes contrasting in drought tolerance (Embrapa 48–tolerant and BR 16–sensitive) were used to study the molecular mechanism underlying this process in soybeans. The hydric potential of the BR 16 reached values of −1.0 and −1.5 MPa, three days before the Embrapa 48, confirming the contrasting drought tolerance. The proteomic, phosphoproteomic and metabolomic profiles were evaluated to detect the metabolic pathways, which were affected by the drought stress. An integrative overview showed that the tolerant plants maintain cell homeostasis and the photosynthetic metabolism was unchanged under the stress condition in contrast to the sensitive genotype that showed several dysregulated pathways. These findings were confirmed by the protein expression and protein regulation by phosphorylation. Furthermore, just small deviations in the metabolic pathways were observed for drought-tolerant plants in comparison to the sensitive genotype. Complex post-translational modification patterns by phosphorylation were detected in some key enzymes, such as carbonic anhydrase, rubisco activase, transketolase and RNA binding proteins during water deficiency. Osmoprotection does not appear to be the major mechanism for tolerance, as indicated by the accumulation of the metabolite and the phytohormone profiles from tolerant and sensitive soybean plants. Thus, regulatory cascades of the metabolic activities, mediated by protein phosphorylation, may cause a higher water use efficiency in the leaves as well as the translocation from the root to the shoot system.

The fungus Pochonia chlamydosporia (Pc) has been increasingly used to control plant-parasitic nematodes. This fungus also increases the uptake of nutrients by plants. However, the mechanisms by which Pc modifies the soil nutrient supply and, or the capacity of plants to acquire nutrients are not fully understood. In this research we investigated possible mechanisms that explain how Pc increases phosphorus uptake by plants. We tested the hypothesis that Pc produces phosphatases, which are enzymes that promote depolymerization of organic phosphate compounds. We further investigated if the known effect of Pc on inorganic phosphate (IP) solubilization could be credited to the release of organic acids and, if so, which organic acids are secreted by the fungus. Three isolates of the fungus (Pc-10, Pc-40, and Pc-46) were used in the study. We found that Pc can produce acid (Pc-10 = 0.0209 U/ml, Pc-40 = 0.0282 U/ml, Pc-46 = 0.0094 U/ml) and alkaline phosphatases (Pc-10 = 0.0490 U/ml, Pc-40 = 0.0000, Pc-46 = 0.0306 U/ml). All isolates were able to solubilize IP. The isolate Pc-10 solubilized higher amounts of phosphate (47.5 mg/l) compared to Pc-40 (37.9 mg/l) and Pc-46 (36.6 mg/l). We also identified three organic acids (acetic, citric, and propionic acid) produced by Pc-10. To our knowledge, this is the first report of Pc producing phosphatases and organic acids.

The data presented here were obtained from the saliva of three triatominae, Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri from Montandon et al. study, doi:10.1016/j.ibmb.2016.02.009 [3]. These data were obtained from spectra generated by the mass spectrometry of proteins observed through the analysis of 2-D electrophoretic profiles. The data were analyzed according to the UniProt code, protein name, protein group, isoelectric point and molecular weight, electrophoretic profile, molecular mass referring to UniProt, volume percentage referring to the spot of the electrophoretic profile, number of peptides and percent coverage found by mass spectrometry related to the particular proteins. In addition, there characterizations made the most significant protein per spot, and also characterizations made for biological processes and molecular functions for all identified proteins. Keywords: Triatomines, Sialoproteomics, Rhodnius prolixus, Triatoma lecticularia, Panstrongylus herreri

Kluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.

Attack by herbivores is a major biotic stress limiting the soybean crop production. Plant defenses against caterpillars include the production of secondary metabolites such as flavonoids, which constitute a diverse group of plant secondary metabolites. Thus, a more discriminate metabolic profiling between genotypes are important for a more comprehensive and reliable characterization of soybean resistance. Therefore, in this study a non-targeted LC/MS-based for analysis of flavonoid profiles of soybean genotypes contrasting to the resistance to A. gemmatalis was applied. Clustering analysis revealed profiles highly distinct between the susceptible UFV 105 AP and the resistant IAC 17 genotypes. This comparative approach enables to identify directly from leaf extract some new compounds related to resistance, some of which were present in higher abundance specifically in the IAC 17 genotype: four Quercetin conjugates, Rutin (Quercetin 3-O-Rutinoside), Quercetin-3,7-O- di-glucoside, Quercetin-3-O-rhamnosylglycoside-7-O-glucoside and Quercetin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside; two Genistein conjugates, Genistein-7-O-diglucoside-dimalonylated and Genistein-7-O-6-O-malonylglucoside; and one Daidzein conjugate, Daidzein-7-O-Glucoside-malonate. The most abundant flavonoid glycoconjugates in soybean leaves belongs to Quercetin and Kaempferol classes. However, only one from the identified compounds was classified as a Kaempferol. The Kaempferol-3-O-L-rhamnopyranosyl-glucopyranoside showed high abundance in the resistant genotype IAC 17. The metabolic profiles generated by LC/MS allowed the reconstruction of the flavonoid biosynthetic pathways, which revealed a constitutive character for herbivory resistance in the resistant genotype IAC-17 and a metabolic regulation for the rechanneling of Quercetin, Kaempferol and Genistein conjugates in soybean. Highest relative abundances were detected for glyconjugates, such as Rutin, Quercetin 3-O-rhamnosylglycoside-7-O-glucoside and Quercitin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside in the leaves of the resistant genotype.

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two-dimensional gel electrophoresis and separation by ion-exchange and reverse-phase high-performance liquid chromatography followed by mass spectrometry using tanden matrix-assisted laser desorption/ionization with time-of-flight (MALDI-TOF/TOF) mass spectrometry and electrospray ionization-quadrupole with time-of-flight (ESI-Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10-ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.

An extracellular β-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce β-glucanase were a glucose concentration of 4 % (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-β-1,3(4)-glucanase. After optimizing the conditions for β-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of β-glucanase in the biotechnological production of soluble β-glucan.

Introduction An acute apical abscess is a severe response of the host to massive invasion of the periapical tissues by bacteria from infected root canals. Although many studies have investigated the microbiota involved in the process, information on the host factors released during abscess formation is scarce. The purpose of this study was to describe the human exoproteome in samples from acute apical abscesses. Methods Fourteen pus samples were obtained by aspiration from patients with an acute apical abscess. Samples were subjected to protein digestion, and the tryptic peptides were analyzed using a mass spectrometer and ion trap instrument. The human proteins identified in this analysis were classified into different functional categories. Results A total of 303 proteins were identified. Most of these proteins were involved in cellular and metabolic processes. Immune system proteins were also very frequent and included immunoglobulins, S100 proteins, complement proteins, and heat shock proteins. Polymorphonuclear neutrophil proteins were also commonly detected, including myeloperoxidases, defensins, elastases, and gelatinases. Iron-sequestering proteins including transferrin and lactoferrin/lactotransferrin were found in many samples. Conclusions The human exoproteome included a wide variety of proteins related to cellular processes, metabolism, and immune response. Proteins involved in different mechanisms against infection, tissue damage, and protection against tissue damage were identified. Knowledge of the presence and function of these proteins using proteomics provides an insight into the complex host-pathogen relationship, the host antimicrobial strategies to fight infections, and the disease pathogenesis.

The binding protein (BiP) has been demonstrated to participate in innate immunity and attenuate endoplasmic reticulum- and osmotic stress-induced cell death. Here, we employed transgenic plants with manipulated levels of BiP to assess whether BiP also controlled developmental and hypersensitive programmed cell death (PCD). Under normal conditions, the BiP-induced transcriptome revealed a robust down-regulation of developmental PCD genes and an up-regulation of the genes involved in hypersensitive PCD triggered by nonhost-pathogen interactions. Accordingly, the BiP-overexpressing line displayed delayed leaf senescence under normal conditions and accelerated hypersensitive response triggered by Pseudomonas syringae pv tomato in soybean (Glycine max) and tobacco (Nicotiana tabacum), as monitored by measuring hallmarks of PCD in plants. The BiP-mediated delay of leaf senescence correlated with the attenuation of N-rich protein (NRP)-mediated cell death signaling and the inhibition of the senescence-associated activation of the unfolded protein response (UPR). By contrast, under biological activation of salicylic acid (SA) signaling and hypersensitive PCD, BiP overexpression further induced NRP-mediated cell death signaling and antagonistically inhibited the UPR. Thus, the SA-mediated induction of NRP cell death signaling occurs via a pathway distinct from UPR. Our data indicate that during the hypersensitive PCD, BiP positively regulates the NRP cell death signaling through a yet undefined mechanism that is activated by SA signaling and related to ER functioning. By contrast, BiP’s negative regulation of leaf senescence may be linked to its capacity to attenuate the UPR activation and NRP cell death signaling. Therefore, BiP can function either as a negative or positive modulator of PCD events.

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.

Drought is the main abiotic stress constraining sugarcane production. However, our limited understanding of the molecular mechanisms involved in the drought stress responses of sugarcane impairs the development of new technologies to increase sugarcane drought tolerance. Here, an integrated approach was performed to reveal the molecular and physiological changes in two closely related sugarcane cultivars, including the most extensively planted cultivar in Brazil (cv. RB867515), in response to moderate (−0.5 MPa) and severe (−1 MPa) drought stress at the transcriptional, translational, and posttranslational levels. The results show common and cultivar exclusive changes in specific genes related to photosynthesis, carbohydrate, amino acid, and phytohormone metabolism. The novel phosphoproteomics and redox proteomic analysis revealed the importance of posttranslational regulation mechanisms during sugarcane drought stress. The shift to soluble sugar, secondary metabolite production, and activation of ROS eliminating processes in response to drought tolerance were mechanisms exclusive to cv. RB867515, helping to explain the better performance and higher production of this cultivar under these stress conditions.

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Bacteria are extraordinarily diverse microorganisms with a huge potential to benefit environmental and agricultural systems. Comprehensive studies in complex habitats such as soils and plants have led to the development of genetic tools to evaluate gene expression and bacterial colonization under controlled or environmental conditions and to obtain genetically engineered organisms for environmental release. In addition, current advances in genomic and metagenomic research will add to the number of genes with potential for biotechnological applications, which will require the development of appropriate genetic systems to fulfill their potential for both industrial and agricultural applications. The aim of the present review is to assess the approaches and recent progress in vector design and genetic tools to study and manipulate plant–bacterial interactions, as well as strategies to construct genetically modified strains for environmental release.

Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

The objective of this work was to produce a polyclonal antiserum against the coat protein (CP) of Papaya lethal yellowing virus (PLYV) and to determine its specificity and sensibility in the diagnosis of the virus, as well as to evaluate the genetic resistance to PLYV in papaya (Carica papaya) accessions and to investigate the capacity of the two-spotted spider mite Tetranychus urticae to acquire and transmit PLYV to the plants. Sixty-five papaya accessions were evaluated. For each accession, ten plants were mechanically inoculated using PLYV-infected plant extracts, and three plants were mock inoculated with phosphate buffer alone and used as negative controls. Ninety days after inoculation, newly-emerging systemic leaves were collected from the inoculated plants, and viral infection was diagnosed by indirect Elisa, using polyclonal antiserum sensible to the in vitro-expressed PLYV CP. Viral transmission by T. urticae was evaluated in greenhouse. The experiments were repeated twice. Polyclonal antiserum recognized the recombinant PLYV CP specifically and discriminated PLYV infection from infections caused by other plant viruses. Out of the 65 papaya accessions evaluated, 15 were considered resistant, 18 moderately resistant, and 32 susceptible. The two-spotted spider mite T. urticae was capable of acquiring PLYV, but not of transmitting it to papaya. O objetivo deste trabalho foi produzir um antissoro policlonal contra a proteína capsidial (PC) do Papaya lethal yellowing virus (PLYV) e determinar sua especificidade e sensibilidade na diagnose do vírus, bem como avaliar a resistência genética de acessos de mamoeiro (Carica papaya) ao PLYV e investigar a capacidade do ácaro rajado Tetranychus urticae em adquirir e transmitir o vírus às plantas. Foram avaliados 65 acessos de mamoeiro. Para cada acesso, dez plantas foram submetidas à inoculação mecânica com extratos de plantas infectadas com PLYV, e três plantas receberam inoculação apenas com tampão de fosfato e foram usadas como controle negativo. Noventa dias após a inoculação, novas folhas sistêmicas emergentes foram coletadas das plantas inoculadas, e a infecção viral foi diagnosticada por Elisa indireto, com uso de antissoro policlonal sensível à PC do PLYV expressa in vitro. A transmissão viral por T. urticae foi avaliada em casa de vegetação. Os experimentos foram repetidos duas vezes. O antissoro policlonal reconheceu a PC do PLYV especificamente e discriminou a infecção pelo PLYV de infecções causadas por outros vírus. Dos 65 acessos de mamoeiros avaliados, 15 foram considerados resistentes, 18 moderadamente resistentes e 32 suscetíveis. O ácaro rajado T. urticae foi capaz de adquirir o PLYV, mas não de transmiti-lo para o mamoeiro.

A modified antibiosis assay was used to evaluate growth inhibition of symbiotic and endophytic bacteria by E. coli strains producing Bacillus amyloliquefaciens ribonuclease, barnase. Inhibition zones were only observed when the assays were performed in minimal medium agar. However, bacterial growth inhibition was not detected when using rich medium or susceptible strains expressing the ribonuclease inhibitor protein, barstar. Our results suggest that barnase may act as a broad range bacteriocin. The ecological significance of these results is discussed.

A new broad-host-range vector expressing constitutively the reporter genes gfp and gusA was used to evaluate nodule occupancy of Phaseolus vulgaris nodules by Rhizobium tropici. The results showed that the pHRGFPGUS plasmid was stably maintained in R. tropici over 45 generations and can therefore be used in nodule competitiveness assays. A new method for determining the nodule occupancy using the green fluorescent protein as a marker is described and is shown to be quick, inexpensive and reliable.

To achieve stable expression of the heterologous and reporter genes in the Bradyrhizobium genome, we constructed suicide plasmids capable of site-directed genomic integration of the gusA, gfp and nifA genes by homologous recombination into non-essential repeated sequences (RS-α), isolated from B. japonicum strain CPAC7 (SEMIA5080). In this report, we describe the strategies to construct the vectors and their use to obtain mutants with site-specific insertions.

Barnase is a potent ribonuclease widely used as a cytotoxic agent, tightly regulated by barstar to maintain cell viability. In this report, we describe a new composite regulatory system to control barnase cytotoxicity and expression, involving barstar and lacI genes under control of the NifA-, σ 54 -dependent Sinorhizobium meliloti nifH promoter, and the barnase gene under control of the LacI-repressible ptac promoter. In this system, expression of the nifH promoter, activated by constitutively expressed NifA, resulted in constitutive synthesis of the LacI and barstar proteins. LacI, in turn, represses transcription of the barnase gene and barstar inhibits any ribonuclease activity. Full expression of the barnase gene induced by IPTG led to cell death. Control of barnase synthesis and activity could be achieved by regulating nifA expression and NifA protein activity by specific environmental signals.

The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif(+) (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif(+) or Nif(-) strains.

Coffee is one of the most valuable agricultural commodities. There is much agronomic research on coffee, but molecular knowledge of its fruit development and ripening is limited. This study reports a comparative proteomic investigation of immature coffee fruits in two early developmental stages: stage 1, cell division and elongation of the perisperm; and stage 2, early growth of the endosperm progressively replacing the perisperm. Proteins were extracted using a modified SDS-phenol method and two-dimensional electrophoresis gels stained with Coomassie blue revealed about 300 well-resolved polypeptide spots in the pH range of 3 to 10. The differentially expressed polypeptides spots were excised, trypsin-digested, and analyzed by MALDI-TOF mass spectrometry. Peptide MS data were searched against the coffee EST database. Most of the identified protein spots are involved in the glycolytic pathway and energy reserve, and are more highly expressed at stage 2.

A novel multienzyme complex, E1 C , and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1 C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1 C and E2 were 21.3 and 27.5 kJ/mol, respectively. The K M for E1 C was 10.25 g/L while for E2 was 6.58 g/L. Both E1 C and E2 were activated by Mn 2+ and CoCl 2 while they were inhibited by SDS, CuSO 4 , FeCl 3 , AgNO 4 , ZnSO 4 and HgCl 2 . E1 C and E2 presented endo-b-1,3–1,4-glucanase activity. E1 C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1 C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A- loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Herbaspirillum spp. are endophytic diazotrophic bacteria associated with important agricultural crops. In this work, we analyzed six strains of H. seropedicae (Z78, M2, ZA69, ZA95, Z152, and Z67) and one strain of H. rubrisubalbicans (M4) by restriction fragment length polymorphism (RFLP) using HindIII or DraI restriction endonucleases, random amplified polymorphic DNA (RAPD), and partial sequencing of 16S rDNA. The results of these analyses ascribed the strains studied to three distinct groups: group I, consisting of M2 and M4; group II, of ZA69; and group III, of ZA95, Z78, Z67, and Z152. RAPD fingerprinting showed a higher variability than the other methods, and each strain had a unique electrophoretic pattern with five of the six primers used. Interestingly, H. seropedicae M2 was found by all analyses to be genetically very close to H. rubrisubalbicans M4. Our results show that RAPD can distinguish between all Herbaspirillum strains tested.