Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Parasites in the neighbourhood: Interactions of the mistletoe Phoradendron affine (Viscaceae) with its dispersers and hosts in urban areas of Brazil

Mistletoes constitute an important food resource for animals in many ecosystems. However, these plants are considered pests in urban areas because of deleterious effects they have on the host trees. Studies in urban areas were mostly focused on listing host species or procedures to control the "pest". In this sense, broader studies including several aspects of mistletoes ecology in urban ecosystems are still missing. We studied the interaction of the mistletoe, Phoradendron affine, with its dispersers and hosts in two urban sites in Uberlandia, Brazil. Phoradendron affine fruits were consumed almost exclusively by Euphonia chlorotica, which was crucial for seed germination. Parasitism was recorded in five hosts, two native (Handroanthus chrysotrichus and Tabebuia roseoalba) and three exotic species (Spathodea campanulata, Ligustrum lucidum and Melia azedarach). Mistletoes were found parasitizing larger host trees, a trend commonly reported for mistletoe-host interaction. Mistletoe seed germination was not affected by the trees species, whether host or non-host, but the radicle of germinated seeds could not penetrate the bark and seedlings invariably died in non-host species. We found a high prevalence of parasitism in our study, in comparison to what previous studies reported for natural areas. The spatial distribution of the hosts and high light incidence on isolated host trees may lead to this high prevalence in urban areas. Rather than eradicated, mistletoes in urban areas should be ecologically managed and their importance for bird species conservation must be considered. More studies to determine which bird species are favoured by mistletoe presence in urban areas will be essential for, this purpose. (C) 2012 Elsevier GmbH. All rights reserved.

Diurnal and nocturnal herbivore induction on maize elicit different innate response of the fall armyworm parasitoid, Campoletis flavicincta

Herbivore-attacked plants produce specific volatile substances that represent important cues for host finding by natural enemies. The fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a voracious herbivore and usually feed on maize in all periods of the day. Given that plant needs light to synthesize de novo herbivore-induced volatiles, volatile blend may be changed depending on time of the day the plant is induced, what could interfere in natural enemy foraging. In this sense, the current study aimed to investigate differential attractiveness of maize elicited by fall armyworm regurgitant under light and dark conditions to its specialist larval parasitoid Campoletis flavicincta (Ashmead) (Hymenoptera: Ichneumonidae). All bioassays were conducted in Y-tube olfactometer to assess parasitoid response to odors from undamaged maize, mechanical damage, and regurgitant-treated plants at 0-1, 5-6, and 24-25 h after induction. The results showed that na < ve wasps were attracted to volatiles emitted by nocturnal regurgitant-treated maize at 5-6 h, but not to odors from diurnal regurgitant-treated plants. The differential attractiveness is likely due to blend composition as nocturnal regurgitant-treated plants emit aromatic compounds and the homoterpene (3E)-4,8-dimethyl-1,3,7-nonatriene in larger amounts than diurnal-treated plants.

Biological and behavioral parameters of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae) are altered by the pathogen Nosema sp (Microsporidia: Nosematidae)

A major issue for mass rearing of insects concerns sanitary conditions and disease. Microsporidian infection (Nosema sp.) in laboratory colonies of Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), used in producing the parasitoid. Cotesia flavipes Cameron (Hymenoptera: Braconidae), is representative of the problems faced by growers and industry. Although C. flavipes has been produced for several years in Brazil for biological control of D. saccharalis, we have only recently observed that the parasitoid becomes infected when developing inside hosts infected with Nosema sp. We assessed the effects of Nosema sp. on C. flavipes, including the ability to locate and select hosts, and evaluated pathogen transmission. Third instar larvae of D. saccharalis were inoculated with Nosema sp. spores at different concentrations and were parasitized when larvae reached fifth instar. Heavily infected D. saccharalis larvae did not support parasitism. Parasitoids that developed in infected D. saccharalis larvae exhibited increased duration of larval and pupal stages, decreased adult longevity and number of offspring, and reduced tibia size compared to parasitoids developing in uninfected D. saccharalis larvae. Infection by Nosema sp. reduced the ability of the C. flavipes parasitoid to distinguish between volatiles released by the sugarcane infested by healthy larvae and pure air. Uninfected parasitoids preferred plants infested with uninfected hosts. But infected C. flavipes did not differentiate between uninfected hosts and those infected with Nosema sp. The pathogen is transmitted from host to parasitoids and parasitoids to hosts. Pathogenic effects of the microsporidium in C. flavipes are sufficiently severe to justify disease management efforts, particularly considering the importance of C. flavipes as a biological control agent in sugarcane. (C) 2012 Elsevier Inc. All rights reserved.

The contribution of a murine CNS-TB model for the understanding of the host-pathogen interactions in the formation of granulomas

Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB, characterized morphologically by brain granulomas and tuberculous meningitis (TBM). Experimental strategies for the study of the host-pathogen interaction through the analysis of granulomas and its intrinsic molecular mechanisms could provide new insights into the neuropathology of TB. To verify whether cerebellar mycobacterial infection induces the main features of the disease in human CNS and better understand the physiological mechanisms underlying the disease, we injected bacillus Calmette-Guerin (BCG) into the mouse cerebellum. BCG-induced CNS-TB is characterized by the formation of granulomas and TBM, a build up of bacterial loads in these lesions, and microglial recruitment into the lesion sites. In addition, there is an enhanced expression of signaling molecules such as nuclear factor-kappa B (NF-kappa B) and there is a presence of inducible nitric oxide synthase (iNOS) in the lesions and surrounding areas. This murine model of cerebellar CNS-TB was characterized by cellular and biochemical immune responses typically found in the human disease. This model could expand our knowledge about granulomas in TB infection of the cerebellum, and help characterize the physiological mechanisms involved with the progression of this serious illness that is responsible for killing millions people every year. (C) 2012 Elsevier B.V. All rights reserved.

Mistletoes Play Different Roles in a Modular Host-Parasite Network

Antagonistic interactions between host plants and mistletoes often form complex networks of interacting species. Adequate characterization of network organization requires a combination of qualitative and quantitative data. Therefore, we assessed the distribution of interactions between mistletoes and hosts in the Brazilian Pantanal and characterized the network structure in relation to nestedness and modularity. Interactions were highly asymmetric, with mistletoes presenting low host specificity (i.e., weak dependence) and with hosts being highly susceptible to mistletoe-specific infections. We found a non-nested and modular pattern of interactions, wherein each mistletoe species interacted with a particular set of host species. Psittacanthus spp. infected more species and individuals and also caused a high number of infections per individual, whereas the other mistletoes showed a more specialized pattern of infection. For this reason, Psittacanthus spp. were regarded as module hubs while the other mistletoe species showed a peripheral role. We hypothesize that this pattern is primarily the result of different seed dispersal systems. Although all mistletoe species in our study are bird dispersed, the frugivorous assemblage of Psittacanthus spp. is composed of a larger suite of birds, whereas Phoradendron are mainly dispersed by Euphonia species. The larger assemblage of bird species dispersing Psittacanthus seeds may also increase the number of hosts colonized and, consequently, its dominance in the study area. Nevertheless, other restrictions on the interactions among species, such as the differential capacity of mistletoe infections, defense strategies of hosts and habitat types, can also generate or enhance the observed pattern.

Fall Armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), Female Moths Respond to Herbivore-Induced Corn Volatiles

In response to herbivore attack, plants release herbivore-induced plant volatiles (HIPVs) that represent important chemical cues for herbivore natural enemies. Additionally, HIPVs have been shown to mediate other ecological interactions with herbivores. Differently from natural enemies that are generally attracted to HIPVs, herbivores can be either attracted or repelled depending on several biological and ecological parameters. Our study aimed to assess the olfactory response of fall armyworm-mated female moths toward odors released by mechanically and herbivore-induced corn at different time intervals. Results showed that female moths strongly respond to corn volatiles, although fresh damaged corn odors (0-1 h) are not recognized by moths. Moreover, females preferred volatiles released by undamaged plant over herbivore-induced plants at 5-6 h. This preference for undamaged plants may reflect an adaptive strategy of moths to avoid competitors and natural enemies for their offspring. We discussed our results based on knowledge about corn volatile release pattern and raise possible explanations for fall armyworm moth behavior.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

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

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

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Endophytic Methylobacterium extorquens expresses a heterologous beta-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions

Abstract Background: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. Results: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. Conclusion: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

“Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions”

Abstract Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities