Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

Natural history of an ant-plant-butterfly interaction in a Neotropical savanna

Non-myrmecophilous lepidopteran larvae using plants bearing ant attractants such as extrafloral nectaries are good models for studying morphological and behavioural mechanisms against ant predation. Udranomia spitzi (Hesperiidae) is a butterfly whose larvae feed on leaves of Ouratea spectabilis (Ochnaceae), a plant with extrafloral nectaries. We described the early stages of U. spitzi, and used field observations and experiments to investigate the defensive strategies of caterpillars against predatory ants. Larvae pass through five instars and pupation occurs inside larval leaf shelters. Ant-exclusion experiments revealed that the presence of ants did not affect significantly caterpillar survival. Predation experiments showed that vulnerability to ant predation decreased with increase in larval size. The present study showed that predatory ants are not as relevant as demonstrated for other systems, and also illustrates how observational data and field experiments can contribute to a better understanding of the biology and ecology of a species of interest.

Where Is My Food? Brazilian Flower Fly Steals Prey from Carnivorous Sundews in a Newly Discovered Plant-Animal Interaction

A new interaction between insects and carnivorous plants is reported from Brazil. Larvae of the predatory flower fly Toxomerus basalis (Diptera: Syrphidae: Syrphinae) have been found scavenging on the sticky leaves of several carnivorous sundew species (Drosera, Droseraceae) in Minas Gerais and São Paulo states, SE Brazil. This syrphid apparently spends its whole larval stage feeding on prey trapped by Drosera leaves. The nature of this plant-animal relationship is discussed, as well as the Drosera species involved, and locations where T. basalis was observed. 180 years after the discovery of this flower fly species, its biology now has been revealed. This is (1) the first record of kleptoparasitism in the Syrphidae, (2) a new larval feeding mode for this family, and (3) the first report of a dipteran that shows a kleptoparasitic relationship with a carnivorous plant with adhesive flypaper traps. The first descriptions of the third instar larva and puparium of T. basalis based on Scanning Electron Microscope analysis are provided.

Monitoramento da expressão gênica de Xanthomonas axonopodis pv. citri em diferentes condições ambientais

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

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.

Management of plant invasions mediated by frugivore interactions

1. Some of the most damaging invasive plants are dispersed by frugivores and this is an area of emerging importance in weed management. It highlights the need for practical information on how frugivores affect weed population dynamics and spread, how frugivore populations are affected by weeds and what management recommendations are available. 2. Fruit traits influence frugivore choice. Fruit size, the presence of an inedible peel, defensive chemistry, crop size and phenology may all be useful traits for consideration in screening and eradication programmes. By considering the effect of these traits on the probability, quality and quantity of seed dispersal, it may be possible to rank invasive species by their desirability to frugivores. Fruit traits can also be manipulated with biocontrol agents. 3. Functional groups of frugivores can be assembled according to broad species groupings, and further refined according to size, gape size, pre- and post-ingestion processing techniques and movement patterns, to predict dispersal and establishment patterns for plant introductions. 4. Landscape fragmentation can increase frugivore dispersal of invasives, as many invasive plants and dispersers readily use disturbed matrix environments and fragment edges. Dispersal to particular landscape features, such as perches and edges, can be manipulated to function as seed sinks if control measures are concentrated in these areas. 5. Where invasive plants comprise part of the diet of native frugivores, there may be a conservation conflict between control of the invasive and maintaining populations of the native frugivore, especially where other threats such as habitat destruction have reduced populations of native fruit species. 6. Synthesis and applications. Development of functional groups of frugivore-dispersed invasive plants and dispersers will enable us to develop predictions for novel dispersal interactions at both population and community scales. Increasingly sophisticated mechanistic seed dispersal models combined with spatially explicit simulations show much promise for providing weed managers with the information they need to develop strategies for surveying, eradicating and managing plant invasions. Possible conservation conflicts mean that understanding the nature of the invasive plant-frugivore interaction is essential for determining appropriate management.

Summer drought alters plant-mediated competition between foliar- and root-feeding insects

Summer droughts are predicted to increase in severity and frequency in the United Kingdom, due to climate change. Few studies have addressed the impacts of drought on interactions between species, and the majority have focussed on increases in CO2 concentration and changes in temperature. Here, the effect of experimental summer drought on the strength of the plant-mediated interaction between leaf-mining Stephensia brunnichella larvae and root-chewing Agriotes larvae was investigated. Agriotes larvae reduced the abundance and performance of S. brunnichella feeding on a mutual host plant, Clinopodium vulgare, as well as the rate of parasitism of the leaf-miner. The interaction did not, however, occur on plants subjected to a severe drought treatment, which were reduced in size. Changes to summer rainfall, due to climate change, may therefore reduce the occurrence of plant-mediated interactions between insect herbivores.

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in 'Valencia' oranges

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serpentine leafminer (Liriomyza trifolii) on potato (Solanum tuberosum): field observations and plant photosynthetic responses to injury

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Size and lipid content of nonmyrmecochorous diaspores: Effects on the interaction with litter-foraging ants in the Atlantic rain forest of Brazil

Ants are often attracted to diaspores not adapted for dispersal by ants. These diaspores may occasionally benefit from this interaction. We selected six nonmyrmecochorous plant species (Virola oleifera, Eugenia stictosepala, Cabralea canjerana, Citharexylum myrianthum, Alchornea glandulosa and Hyeronima alchorneoides) whose diaspores differ in size and lipid content, and investigated how these features affect the outcome of ant-diaspore interactions on the floor of a lowland Atlantic forest of Southeast Brazil. A total of 23 ant species were seen interacting with diaspores on the forest floor. Ants were generally rapid at discovering and cleaning the diaspore pulp or aril. Recruitment rate and ant attendance were higher for lipid-rich diaspores than for lipid-poor ones. Removal rate and displacement distance were higher for small diaspores. The large ponerine ant Pachycondyla striata, one of the most frequent attendants to lipid-rich arillate diaspores, transported the latter into their nests and discarded clean intact seeds on refuse piles outside the nest. Germination tests with cleaned and uncleaned diaspores revealed that the removal of pulp or aril may increase germination success in Virola oleifera, Cabralea canjerana, Citharexylum myrianthum and Alchornea glandulosa. Gas chromatography analyses revealed a close similarity in the fatty acid composition of the arils of the lipid-rich diaspores and the elaiosome of a typical myrmecochorous seed (Ricinus communis), corroborating the suggestion that some arils and elaiosomes are chemically similar. Although ant-derived benefits to diaspores - secondary dispersal and/or increased germination - varied among the six plant species studied, the results enhanced the role of ant-diaspore interactions in the post-dispersal fates of nonmyrmecochorous seeds in tropical forests. The size and the lipid-content of the diaspores were shown to be major determinants of the outcome of such interactions.

The role of multiple partners in a digestive mutualism with a protocarnivorous plant

Background and aimsThe protocarnivorous plant Paepalanthus bromelioides (Eriocaulaceae) is similar to bromeliads in that this plant has a rosette-like structure that allows rainwater to accumulate in leaf axils (i.e. phytotelmata). Although the rosettes of P. bromelioides are commonly inhabited by predators (e.g. spiders), their roots are wrapped by a cylindrical termite mound that grows beneath the rosette. In this study it is predicted that these plants can derive nutrients from recycling processes carried out by termites and from predation events that take place inside the rosette. It is also predicted that bacteria living in phytotelmata can accelerate nutrient cycling derived from predators.MethodsThe predictions were tested by surveying plants and animals, and also by performing field experiments in rocky fields from Serra do Cipó, Brazil, using natural abundance and enriched isotopes of 15N. Laboratory bioassays were also conducted to test proteolytic activities of bacteria from P. bromelioides rosettes.Key ResultsAnalyses of 15N in natural nitrogen abundances showed that the isotopic signature of P. bromelioides is similar to that of carnivorous plants and higher than that of non-carnivorous plants in the study area. Linear mixing models showed that predatory activities on the rosettes (i.e. spider faeces and prey carcass) resulted in overall nitrogen contributions of 26·5 % (a top-down flux). Although nitrogen flux was not detected from termites to plants via decomposition of labelled cardboard, the data on 15N in natural nitrogen abundance indicated that 67 % of nitrogen from P. bromelioides is derived from termites (a bottom-up flux). Bacteria did not affect nutrient cycling or nitrogen uptake from prey carcasses and spider faeces.ConclusionsThe results suggest that P. bromelioides derive nitrogen from associated predators and termites, despite differences in nitrogen cycling velocities, which seem to have been higher in nitrogen derived from predators (leaves) than from termites (roots). This is the first study that demonstrates partitioning effects from multiple partners in a digestion-based mutualism. Despite most of the nitrogen being absorbed through their roots (via termites), P. bromelioides has all the attributes necessary to be considered as a carnivorous plant in the context of digestive mutualism. © 2012 The Author. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

The role of prophage in plant-pathogenic bacteria

A diverse set of phage lineages is associated with the bacterial plant-pathogen genomes sequenced to date. Analysis of 37 genomes revealed 5,169 potential genes (approximately 4.3 Mbp) of phage origin, and at least 50 had no function assigned or are nonessential to phage biology. Some phytopathogens have transcriptionally active prophage genes under conditions that mimic plant infection, suggesting an association between plant disease and prophage transcriptional modulation. The role of prophages within genomes for cell biology varies. For pathogens such as Pectobacterium, Pseudomonas, Ralstonia, and Streptomyces, involvement of prophage in disease symptoms has been demonstrated. In Xylella and Xanthomonas, prophage activity is associated with genome rearrangements and strain differentiation. For other pathogens, prophage roles are yet to be established. This review integrates available information in a unique interface (http://propnav.esalq.usp.br) that may be assessed to improve research in prophage biology and its association with genome evolution and pathogenicity. © Copyright ©2013 by Annual Reviews. All rights reserved.

Transcriptoma (RNA-seq) de laranja doce Valência (Citrus sinensis L. Osbeck) e Kumquat (Fortunella spp.) infectadas por Xanthomonas citri subsp. citri

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR