Microsatellite markers in analysis of resistance to coffee leaf miner in Arabica coffee

The objective of this work was to analyze coffee (Coffea arabica) genotypes resistant to the coffee leaf miner (Leucoptera coffeella) using microsatellite markers. Sixty-six loci were evaluated, of which 63 were obtained from the Brazilian Coffee Expressed Sequence Tag (EST) database. These loci were amplified in bulks of individuals from F5 progenies of 'Siriema' (C. arabica x C. racemosa) resistant and susceptible to the insect, in eight samples of C. racemosa, and in a F6 population of 'Siriema' with 91 individuals segregating for resistance to the leaf miner. Polymorphisms were verified for two simple sequence repeat (SSR) loci in bulks of the susceptible progenies. The two polymorphic alleles were present in around 70% of the susceptible genotypes in F5 and in approximately 90% of the susceptible individuals in F6. However, the polymorphic EST-SSR markers among populations contrasting for resistance to leaf miner were not correlated to the evaluated characteristics. SSR markers show inter- and intraspecific polymorphism in C. arabica and C. racemosa.

Odorant binding proteins of the red imported fire ant, Solenopsis invicta: an example of the problems facing the analysis of widely divergent proteins.

We describe the odorant binding proteins (OBPs) of the red imported fire ant, Solenopsis invicta, obtained from analyses of an EST library and separate 454 sequencing runs of two normalized cDNA libraries. We identified a total of 18 putative functional OBPs in this ant. A third of the fire ant OBPs are orthologs to honey bee OBPs. Another third of the OBPs belong to a lineage-specific expansion, which is a common feature of insect OBP evolution. Like other OBPs, the different fire ant OBPs share little sequence similarity (∼ 20%), rendering evolutionary analyses difficult. We discuss the resulting problems with sequence alignment, phylogenetic analysis, and tests of selection. As previously suggested, our results underscore the importance for careful exploration of the sensitivity to the effects of alignment methods for data comprising widely divergent sequences.

Transcriptome analysis of intraspecific competition in Arabidopsis thaliana reveals organ-specific signatures related to nutrient acquisition and general stress response pathways.

ABSTRACT: BACKGROUND: Plants are sessile and therefore have to perceive and adjust to changes in their environment. The presence of neighbours leads to a competitive situation where resources and space will be limited. Complex adaptive responses to such situation are poorly understood at the molecular level. RESULTS: Using microarrays, we analysed whole-genome expression changes in Arabidopsis thaliana plants subjected to intraspecific competition. The leaf and root transcriptome was strongly altered by competition. Differentially expressed genes were enriched in genes involved in nutrient deficiency (mainly N, P, K), perception of light quality, and responses to abiotic and biotic stresses. Interestingly, performance of the generalist insect Spodoptera littoralis on densely grown plants was significantly reduced, suggesting that plants under competition display enhanced resistance to herbivory. CONCLUSIONS: This study provides a comprehensive list of genes whose expression is affected by intraspecific competition in Arabidopsis. The outcome is a unique response that involves genes related to light, nutrient deficiency, abiotic stress, and defence responses.

Recognition of insect eggs in Arabidopsis modulates defense responses

Plants activate direct and indirect defenses in response to insect egg deposition. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. Even though this innate immune response did not affect egg survival in Arabidopsis, we could show that different insect eggs elicit specific gene expression changes. Additionally, egg- induced necrosis could be observed in a variety of plants from different families ranging from dicotyledonous plants to monocots, suggesting that insect-egg detection by plants is a widespread mechanism and that different insect species contain elicitors of immune responses. Extracts from caterpillars and eggs contain elicitors that co-purified over several extraction steps. Chemical fractionation of caterpillar extracts lead to the characterisation of an active compound that was determined to be a triglyceride by NMR analysis. The exact structure of the side chains as well as the elicitor's presence in insect eggs have yet to be confirmed.We also found that the plant defense signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls the defense against fungal and bacterial pathogens whereas it negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defense against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defense. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not found in the SA-deficient mutant sid2-l, indicating that SA mediates this phenomenon. These data reveal an intriguing facet of the crosstalk between SA- and JA-signalling pathways and suggest that insects have evolved a way to suppress the induction of defense genes by laying eggs that release elicitors. Additionally, we demonstrated that mutants of known crosstalk regulators, including nprl-1, tga2356, ein2-l and wrky70-l, are not affected in egg-induced suppression of herbivore defenses. JA treatment was not able to alleviate this SA/JA negative crosstalk, suggesting that this suppression operates through a novel mechanism downstream of JA biosynthesis.

Arabidopsis Basic Helix-Loop-Helix Transcription Factors MYC2, MYC3, and MYC4 Regulate Glucosinolate Biosynthesis, Insect Performance, and Feeding Behavior.

Arabidopsis thaliana plants fend off insect attack by constitutive and inducible production of toxic metabolites, such as glucosinolates (GSs). A triple mutant lacking MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that are known to additively control jasmonate-related defense responses, was shown to have a highly reduced expression of GS biosynthesis genes. The myc2 myc3 myc4 (myc234) triple mutant was almost completely devoid of GS and was extremely susceptible to the generalist herbivore Spodoptera littoralis. On the contrary, the specialist Pieris brassicae was unaffected by the presence of GS and preferred to feed on wild-type plants. In addition, lack of GS in myc234 drastically modified S. littoralis feeding behavior. Surprisingly, the expression of MYB factors known to regulate GS biosynthesis genes was not altered in myc234, suggesting that MYC2/MYC3/MYC4 are necessary for direct transcriptional activation of GS biosynthesis genes. To support this, chromatin immunoprecipitation analysis showed that MYC2 binds directly to the promoter of several GS biosynthesis genes in vivo. Furthermore, yeast two-hybrid and pull-down experiments indicated that MYC2/MYC3/MYC4 interact directly with GS-related MYBs. This specific MYC-MYB interaction plays a crucial role in the regulation of defense secondary metabolite production and underlines the importance of GS in shaping plant interactions with adapted and nonadapted herbivores.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Diverse set of Turing nanopatterns coat corneae across insect lineages.

Nipple-like nanostructures covering the corneal surfaces of moths, butterflies, and Drosophila have been studied by electron and atomic force microscopy, and their antireflective properties have been described. In contrast, corneal nanostructures of the majority of other insect orders have either been unexamined or examined by methods that did not allow precise morphological characterization. Here we provide a comprehensive analysis of corneal surfaces in 23 insect orders, revealing a rich diversity of insect corneal nanocoatings. These nanocoatings are categorized into four major morphological patterns and various transitions between them, many, to our knowledge, never described before. Remarkably, this unexpectedly diverse range of the corneal nanostructures replicates the complete set of Turing patterns, thus likely being a result of processes similar to those modeled by Alan Turing in his famous reaction-diffusion system. These findings reveal a beautiful diversity of insect corneal nanostructures and shed light on their molecular origin and evolutionary diversification. They may also be the first-ever biological example of Turing nanopatterns.

RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.

Predicting richness and composition in mountain insect communities at high resolution: a new test of the SESAM framework

Aim The aim of this study was to test different modelling approaches, including a new framework, for predicting the spatial distribution of richness and composition of two insect groups. Location The western Swiss Alps. Methods We compared two community modelling approaches: the classical method of stacking binary prediction obtained fromindividual species distribution models (binary stacked species distribution models, bS-SDMs), and various implementations of a recent framework (spatially explicit species assemblage modelling, SESAM) based on four steps that integrate the different drivers of the assembly process in a unique modelling procedure. We used: (1) five methods to create bS-SDM predictions; (2) two approaches for predicting species richness, by summing individual SDM probabilities or by modelling the number of species (i.e. richness) directly; and (3) five different biotic rules based either on ranking probabilities from SDMs or on community co-occurrence patterns. Combining these various options resulted in 47 implementations for each taxon. Results Species richness of the two taxonomic groups was predicted with good accuracy overall, and in most cases bS-SDM did not produce a biased prediction exceeding the actual number of species in each unit. In the prediction of community composition bS-SDM often also yielded the best evaluation score. In the case of poor performance of bS-SDM (i.e. when bS-SDM overestimated the prediction of richness) the SESAM framework improved predictions of species composition. Main conclusions Our results differed from previous findings using community-level models. First, we show that overprediction of richness by bS-SDM is not a general rule, thus highlighting the relevance of producing good individual SDMs to capture the ecological filters that are important for the assembly process. Second, we confirm the potential of SESAM when richness is overpredicted by bS-SDM; limiting the number of species for each unit and applying biotic rules (here using the ranking of SDM probabilities) can improve predictions of species composition

Study of tergal glands morphogenesis through an integrative analysis of genomic data

About 50% of living species are holometabolan insects. Therefore, unraveling the ori- gin of insect metamorphosis from the hemimetabolan (gradual metamorphosis) to the holometabolan (sudden metamorphosis at the end of the life cycle) mode is equivalent to explaining how all this biodiversity originated. One of the problems with studying the evolution from hemimetaboly to holometaboly is that most information is available only in holometabolan species. Within the hemimetabolan group, our model, the cock- roach Blattella germanica, is the most studied species. However, given that the study of adult morphogenesis at organismic level is still complex, we focused on the study of the tergal gland (TG) as a minimal model of metamorphosis. The TG is formed in tergites 7 and 8 (T7-8) in the last days of the last nymphal instar (nymph 6). The comparative study of four T7-T8 transcriptomes provided us with crucial keys of TG formation, but also essential information about the mechanisms and circuitry that allows the shift from nymphal to adult morphogenesis.

Geostical analysis of spatial dynamics of Citrus Variegated Chlorossis

The spatial dynamics of Citrus Variegated Chlorosis (CVC) was studied in a five-year old commercial orchard of 'Valencia' sweet orange (Citrus sp.) trees, located in the northern region of the state of São Paulo, Brazil. One thousand trees were assessed in 25 rows of 40 trees, planted at 8 x 5 m spacing. Disease incidence data were taken beginning in March 1994 and ending in January 1996, at intervals of four to five months. Disease aggregation was observed through the dispersion index analysis (Ib), which was calculated by dividing the area into quadrants. CVC spatial dynamics was examined using semivariogram analysis, which revealed that the disease was aggregated in the field forming foci of 10 to 14 m. For each well-fitted model, a kriging map was created to better visualize the distribution of the disease. The spherical model was the best fit for the data in this study. Kriging maps also revealed that the incidence of CVC increased in periods during which the trees underwent vegetative growth, coinciding with greater expected occurrence of insect vectors of the bacterium in the field.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Plant rhizosphere specificity and variability in the insect and plant adhesins, Madl and Mad2, within the genus Metarhizium suggest plant adaptation as an evolutionary force

Metarhizium is a soil-inhabiting fungus currently used as a biological control agent against various insect species, and research efforts are typically focused on its ability to kill insects. In section 1, we tested the hypothesis that species of Metarhizium are not randomly distributed in soils but show plant rhizosphere-specific associations. Results indicated an association of three Metarhizium species (Metarhizium robertsii, M. brunneum and M. guizhouense) with the rhizosphere of certain types of plant species. M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense, which was supported by in vitro experiments with grass root exudate. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types. In section 2, we explored the variation in the insect adhesin, Madl, and the plant adhesin, Mad2, in fourteen isolates of Metarhizium representing seven different species. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions. Phylogenetic analysis of 5' EF-Ia, which is used for species identification, as well as Madl and Mad2 sequences demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1a than Madl. This suggests Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation. While other abiotic and biotic factors cannot be excluded in contributing to divergence, it appears that plant associations have been the driving factor causing divergence among Metarhizium species.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.