Possible Host Adaptation as an Evolution Factor of Cowpea aphid-borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid-borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C.hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous-infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea-infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.

A tritrophic signal that attracts parasitoids to host-damaged plants withstands disruption by non-host herbivores

Background: Volatiles emitted by herbivore-infested plants are highly attractive to parasitoids and therefore have been proposed to be part of an indirect plant defense strategy. However, this proposed function of the plant-provided signals remains controversial, and it is unclear how specific and reliable the signals are under natural conditions with simultaneous feeding by multiple herbivores. Phloem feeders in particular are assumed to interfere with plant defense responses. Therefore, we investigated how attack by the piercing-sucking cicadellid Euscelidius variegatus influences signaling by maize plants in response to the chewing herbivore Spodoptera littoralis.Results: The parasitoid Cotesia marginiventris strongly preferred volatiles of plants infested with its host S. littoralis. Overall, the volatile emissions induced by S. littoralis and E. variegatus were similar, but higher levels of certain wound-released compounds may have allowed the wasps to specifically recognize plants infested by hosts. Expression levels of defense marker genes and further behavioral bioassays with the parasitoid showed that neither the physiological defense responses nor the attractiveness of S. littoralis infested plants were altered by simultaneous E. variegatus attack.Conclusions: Our findings imply that plant defense responses to herbivory can be more robust than generally assumed and that ensuing volatiles convey specific information about the type of herbivore that is attacking a plant, even in complex situations with multiple herbivores. Hence, the results of this study support the notion that herbivore-induced plant volatiles may be part of a plant's indirect defense stratagem. © 2010 Erb et al; licensee BioMed Central Ltd.

Higher plant diversity promotes higher diversity of fungal pathogens, while it decreases pathogen infection per plant

Fungal plant pathogens are common in natural communities where they affect plant physiology, plant survival, and biomass production. Conversely, pathogen transmission and infection may be regulated by plant community characteristics such as plant species diversity and functional composition that favor pathogen diversity through increases in host diversity while simultaneously reducing pathogen infection via increased variability in host density and spatial heterogeneity. Therefore, a comprehensive understanding of multi-host multi-pathogen interactions is of high significance in the context of biodiversity-ecosystem functioning. We investigated the relationship between plant diversity and aboveground obligate parasitic fungal pathogen (''pathogens'' hereafter) diversity and infection in grasslands of a long-term, large-scale, biodiversity experiment with varying plant species (1-60 species) and plant functional group diversity (1-4 groups). To estimate pathogen infection of the plant communities, we visually assessed pathogen-group presence (i.e., rusts, powdery mildews, downy mildews, smuts, and leaf-spot diseases) and overall infection levels (combining incidence and severity of each pathogen group) in 82 experimental plots on all aboveground organs of all plant species per plot during four surveys in 2006. Pathogen diversity, assessed as the cumulative number of pathogen groups on all plant species per plot, increased log-linearly with plant species diversity. However, pathogen incidence and severity, and hence overall infection, decreased with increasing plant species diversity. In addition, co-infection of plant individuals by two or more pathogen groups was less likely with increasing plant community diversity. We conclude that plant community diversity promotes pathogen-community diversity while at the same time reducing pathogen infection levels of plant individuals.

Salicylic Acid, a Plant Defense Hormone, Is Specifically Secreted by a Molluscan Herbivore

Slugs and snails are important herbivores in many ecosystems. They differ from other herbivores by their characteristic mucus trail. As the mucus is secreted at the interface between the plants and the herbivores, its chemical composition may play an essential role in plant responses to slug and snail attack. Based on our current knowledge about host-manipulation strategies employed by pathogens and insects, we hypothesized that mollusks may excrete phytohormone-like substances into their mucus. We therefore screened locomotion mucus from thirteen molluscan herbivores for the presence of the plant defense hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). We found that the locomotion mucus of one slug, Deroceras reticulatum, contained significant amounts of SA, a plant hormone that is known to induce resistance to pathogens and to suppress plant immunity against herbivores. None of the other slugs and snails contained SA or any other hormone in their locomotion mucus. When the mucus of D. reticulatum was applied to wounded leaves of A. thaliana, the promotor of the SA-responsive gene pathogenesis related 1 (PR1) was activated, demonstrating the potential of the mucus to regulate plant defenses. We discuss the potential ecological, agricultural and medical implications of this finding.

The Role of Plant Primary and Secondary Metabolites in Root-Herbivore Behaviour, Nutrition and Physiology

Many insect herbivores feed on belowground plant tissues. In this chapter, we discuss how they have adapted to deal with root primary and secondary metabolites. It is becoming evident that root herbivores can use root volatiles and exudates for host location and foraging. Their complex sensory apparatus suggests a sophisticated recognition and signal transduction system. Furthermore, endogenous metabolites trigger attractive or repellent responses in root feeders, indicating that they may specifically fine-tune food uptake to meet their dietary needs. Little evidence for direct toxic effects of root secondary metabolites has accumulated so far, indicating high prevalence of tolerance mechanisms. Root herbivores furthermore facilitate the entry of soil microbes into the roots, which may influence root nutritional quality. Investigating the role of plant metabolites in an ecologically and physiologically relevant context will be crucial to refine our current models on root-herbivore physiology and behaviour in the future.

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against l-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His 6 -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1 D299A non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.

Genomics of host specificity in the Rhizobium-legume simbiosis

Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus. However, previous evidence suggests that some genotypes are more adapted to a given plant host than others, and that the plant host can select specific genotypes among those present in a given soil population. We have used a population genomics approach to confirm that this is indeed the case, and to analyze the specific genotypic characteristics that each plant host selects

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.