RESUMO
IQ Domain 1 (IQD1) is a novel Arabidopsis thaliana calmodulin-binding protein, which was found to be a positive regulator of glucosinolate (GS) accumulation and plant defense responses against insects. We demonstrate here that the IQD1 overexpressing line (IQD1 OXP ) was also more resistant also to the necrotrophic fungus Botrytis cinerea, whereas an IQD1 knockout line (iqd1-1) was much more sensitive. Furthermore, we showed that IQD1 is up-regulated by jasmonic acid (JA) and downregulated by salicylic acid (SA). A comparison of whole transcriptome expression between iqd1-1 and wild type plants revealed a substantial downregulation of genes involved in plant defense and hormone regulation. Further examination revealed a marked reduction of SA and increases in the levels of ethylene, JA and abscisic acid response genes in the iqd1-1 line. Moreover, quantification of SA, JA, and abscisic acids in IQD1 OXP and iqd1-1 lines relative to the wild type, showed a significant reduction in endogenous JA levels in the knockout line, simultaneously with increased SA levels. Relations between IQD1 OXP and mutants defective in plant-hormone response indicated that IQD1 cannot rescue the absence of NPR1 or impaired SA accumulation in the NahG line. IQD1 cannot rescue ein2 or eto1 mutations connected to the ethylene pathway involved in both defense responses against B. cinerea and in regulating GS accumulation. Furthermore, IQD1cannot rescue the aos, coi1 or jar1mutations, all involved in the defense response against B. cinerea and it depends on JAR1 to control indole glucosinolate accumulation. We also found that in the B. cinerea, which infected the iqd1-1 mutant, the most abundant upregulated group of proteins is involved in the degradation of complex carbohydrates, as correlated with the sensitivity of this mutant. In summary, our results suggest that IQD1 is an important A. thaliana defensive protein against B. cinerea that is integrated into several important pathways, such as those involved in plant defense and hormone responses.
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Beneficial microorganisms need to overcome the plant defense system to establish on or within plant tissues. Like pathogens, beneficial microbes can manipulate a plant's immunity pathways, first by suppressing and hiding to establish on the host and then by inducing resistance to protect the plant. In the current study, we demonstrated that although Pseudozyma aphidis can activate microbe-associated molecular pattern (MAMP)-associated genes, it does not activate MAMP-triggered callose deposition and can, moreover, suppress such deposition triggered by Flg22 or chitin. While MAMP-associated gene activation by P. aphidis was not dependent on salicylic acid, jasmonic acid, or ethylene signaling, suppression of MAMP-triggered callose deposition required the salicylic acid and jasmonic acid signaling factors JAR1-1 and E3 ubiquitin ligase COI1 yet did not rely on EIN2, NPR1, or the transcription factor JIN1/MYC2. We also demonstrated the ability of P. aphidis, known to be an epiphytic yeast-like organism, to penetrate the stomata and establish within plant tissues, as do endophytes. These results thus demonstrate the potential of P. aphidis to suppress MAMP-elicited defenses in order to establish on and within host plant tissues. IMPORTANCE Our study demonstrates the ability of P. aphidis to penetrate into plant tissues, where it avoids and overcomes plant defense systems in order to establish and subsequently protect the plant.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Basidiomycota , Glucanos , Doenças das Plantas , Folhas de Planta/metabolismo , Ácido Salicílico/metabolismoRESUMO
Plant pathogens challenge our efforts to maximize crop production due to their ability to rapidly develop resistance to pesticides. Fungal biocontrol agents have become an important alternative to chemical fungicides, due to environmental concerns related to the latter. Here we review the complex modes of action of biocontrol agents in general and epiphytic yeasts belonging to the genus Pseudozyma specifically and P. aphidis in particular. Biocontrol agents act through multiple direct and indirect mechanisms, which are mainly based on their secretions. We discuss the direct modes of action, such as antibiosis, reactive oxygen species-producing, and cell wall-degrading enzyme secretions which can also play a role in mycoparasitism. In addition, we discuss indirect modes of action, such as hyperbiotrophy, induced resistance and growth promotion based on the secretion of effectors and elicitors from the biocontrol agent. Due to their unique characteristics, epiphytic yeasts hold great potential for use as biocontrol agents, which may be more environmentally friendly than conventional pesticides and provide a way to reduce our dependency on fungicides based on increasingly expensive fossil fuels. No less important, the complex mode of action of Pseudozyma-based biocontrol agents can also reduce the frequency of resistance developed by pathogens to these agents.
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Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.
Assuntos
Arabidopsis/microbiologia , Botrytis/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Glucosinolatos/química , DNA Complementar , DNA Fúngico , Deleção de Genes , Mutação , Doenças das Plantas/microbiologia , RNA Fúngico , Saccharomyces cerevisiae/metabolismoRESUMO
Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.
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Botrytis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ustilaginales/patogenicidade , Apoptose/fisiologia , Micélio/metabolismoRESUMO
Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.
Assuntos
Antioxidantes/metabolismo , Betalaínas/biossíntese , Produtos Agrícolas/genética , Pigmentação/genética , Vias Biossintéticas/genética , Botrytis/fisiologia , Cor , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Resistência à Doença/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Solanum melongena/genética , Solanum melongena/metabolismo , Solanum melongena/microbiologia , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ-induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense-regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase-associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.
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Epiphytic yeasts, which colonize plant surfaces, may possess activity that can be harnessed to help plants defend themselves against various pathogens. Due to their unique characteristics, epiphytic yeasts belonging to the genus Pseudozyma hold great potential for use as biocontrol agents. We identified a unique, biologically active isolate of the epiphytic yeast Pseudozyma aphidis that is capable of inhibiting Botrytis cinerea via a dual mode of action, namely induced resistance and antibiosis. Here, we show that strain L12 of P. aphidis can reduce the severity of powdery mildew caused by Podosphaera xanthii on cucumber plants with an efficacy of 75%. Confocal and scanning electron microscopy analyses demonstrated P. aphidis proliferation on infected tissue and its production of long hyphae that parasitize the powdery mildew hyphae and spores as an ectoparasite. We also show that crude extract of P. aphidis metabolites can inhibit P. xanthii spore germination in planta. Our results suggest that in addition to its antibiosis as mode of action, P. aphidis may also act as an ectoparasite on P. xanthii. These results indicate that P. aphidis strain L12 has the potential to control powdery mildew.
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The ability of plant pathogens to rapidly develop resistance to commonly used pesticides challenges efforts to maximize crop production. Fungal biocontrol agents have become an important alternative to chemical fungicides as a result of environmental concerns regarding conventional pesticides, including resistance issues. The complex mode of action of biocontrol agents reduces the likelihood that pathogens will develop resistance to them. We recently isolated a unique, biologically active isolate of the epiphytic fungus Pseudozyma aphidis. We show that the extracellular metabolites secreted by our P. aphidis isolate can inhibit Xanthomonas campestris pv. vesicatoria, X. campestris pv. campestris, Pseudomonas syringae pv. tomato, Erwinia amylovora, Clavibacter michiganensis, and Agrobacterium tumefaciens in vitro. Moreover, application of Pseudozyma aphidis spores on tomato plants in the greenhouse significantly reduced (by 60%) the incidence of bacterial wilt and canker disease caused by C. michiganensis subsp. michiganensis on those plants as well as disease severity by 35%. Furthermore, infected plants treated with P. aphidis were 25% taller than control infected plants. We found that P. aphidis activates PR1a-and other pathogenesis-related genes in tomato plants-and can trigger an induced-resistance response against C. michiganensis that proceeds in a salicylic-acid-independent manner, as shown using NahG-transgenic tomato plants.
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The cuticle plays an important role in plant interactions with pathogens and with their surroundings. The cuticle acts as both a physical barrier against physical stresses and pathogens and a chemical deterrent and activator of the plant defense response. Cuticle production in tomato plants is regulated by several transcription factors, including SlSHINE3, an ortholog of the Arabidopsis WIN/SHN3. Here we used a SlSHINE3-overexpressing (SlSHN3-OE) and silenced (Slshn3-RNAi) lines and a mutant in SlCYP86A69 (Slcyp86A69)--a direct target of SlSHN3--to analyze the roles of the leaf cuticle and cutin content and composition in the tomato plant's defense response to the necrotrophic foliar pathogen Botrytis cinerea and the biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria. We showed that SlSHN3, which is predominantly expressed in tomato fruit epidermis, also affects tomato leaf cuticle, as morphological alterations in the SlSHN3-OE leaf tissue resulted in shiny, stunted and permeable leaves. SlSHN3-OE leaves accumulated 38% more cutin monomers than wild-type leaves, while Slshn3-RNAi and Slcyp86A69 plants showed a 40 and 70% decrease in leaf cutin monomers, respectively. Overexpression of SlSHN3 resulted in resistance to B. cinerea infection and to X. campestris pv. vesicatoria, correlated with cuticle permeability and elevated expression of pathogenesis-related genes PR1a and AOS. Further analysis revealed that B. cinerea-infected Slshn3-RNAi plants are more sensitive to B. cinerea and produce more hydrogen peroxide than wild-type plants. Cutin monomer content and composition differed between SlSHN3-OE, Slcyp86A69, Slshn3-RNAi and wild-type plants, and cutin monomer extracted from SlSHN3-OE plants altered the expression of pathogenesis-related genes in wild-type plants.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Fatores de Transcrição/metabolismo , Apoptose , Solanum lycopersicum/genética , Mutação , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Xanthomonas campestrisRESUMO
Sweetpotato is a nutritional source worldwide. Soft rot caused by Rhizopus spp. is a major limiting factor in the storage of produce, rendering it potentially unsafe for human consumption. In this study, Rhizopus oryzae was used to develop a concept of postharvest disease control by weakening the pathogen through induction of spore germination under starvation conditions. We isolated the sweetpotato active fractions (SPAFs) that induce spore germination and used them at a low dose to enhance spore weakening caused by starvation. Germination in SPAF at 1 mg/ml weakened the pathogen spores by delaying their ability to form colonies on rich media and by increasing their sensitivity to heat stress. The weakening effect was also supported by reduced metabolic activity, as detected by Alarmar Blue fluorescent dye assays. Spores incubated with SPAF at 1 mg/ml showed DNA fragmentation in some of their nuclei, as observed by TUNEL assay. In addition, these spores exhibited changes in ultrastructural morphology (i.e., shrinkage of germ tubes, nucleus deformation, and vacuole formation) which are hallmarks of programmed cell death. We suggest that induction of spore germination under starvation conditions increases their susceptibility to stress and, therefore, might be considered a new strategy for pathogen control.
Assuntos
Ipomoea batatas/química , Doenças das Plantas/microbiologia , Extratos Vegetais/farmacologia , Rhizopus/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Apoptose , Fragmentação do DNA , Relação Dose-Resposta a Droga , Temperatura Alta , Ipomoea batatas/microbiologia , Extratos Vegetais/isolamento & purificação , Rhizopus/citologia , Rhizopus/genética , Rhizopus/fisiologia , Esporos Fúngicos/fisiologiaRESUMO
Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.
Assuntos
Alternaria/fisiologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Glucosinolatos/metabolismo , Doenças das Plantas/microbiologia , Arabidopsis/metabolismo , Resistência à Doença , Interações Hospedeiro-Patógeno , Hidrólise , Indóis/metabolismo , Tiazóis/metabolismoRESUMO
The plant cell cuticle serves as the first barrier protecting plants from mechanical injury and invading pathogens. The cuticle can be breached by cutinase-producing pathogens and the degradation products may activate pathogenesis signals in the invading pathogens. Cuticle degradation products may also trigger the plant's defense responses. Botrytis cinerea is an important plant pathogen, capable of attacking and causing disease in a wide range of plant species. Arabidopsis thaliana shn1-1D is a gain-of-function mutant, which has a modified cuticular lipid composition. We used this mutant to examine the effect of altering the whole-cuticle metabolic pathway on plant responses to B. cinerea attack. Following infection with B. cinerea, the shn1-1D mutant discolored more quickly, accumulated more H2O2, and showed accelerated cell death relative to wild-type (WT) plants. Whole transcriptome analysis of B. cinerea-inoculated shn1-1D vs. WT plants revealed marked upregulation of genes associated with senescence, oxidative stress and defense responses on the one hand, and genes involved in the magnitude of defense-response control on the other. We propose that altered cutin monomer content and composition of shn1-1D plants triggers excessive reactive oxygen species accumulation and release which leads to a strong, unique and uncontrollable defense response, resulting in plant sensitivity and death.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Resistência à Doença/genética , Expressão Gênica , Doenças das Plantas/genética , Transativadores/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Arabidopsis/microbiologia , Bactérias , Morte Celular/genética , Fungos , Peróxido de Hidrogênio/metabolismo , Mutação , Oxirredução , Estresse Oxidativo/genética , Fenótipo , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Species of the epiphytic fungus Pseudozyma are not pathogenic to plants and can be used as biocontrol agents against plant pathogens. Deciphering how they induce plant defense might contribute to their use for plant protection and expand our understanding of molecular plant-pathogen interactions. Here we show that Pseudozyma aphidis isolate L12, which is known to induce jasmonic acid- and salicylic acid-independent systemic resistance, can also activate local and systemic resistance in an ethylene-independent manner. We also show that P. aphidis localizes exclusively to the surface of the plant leaf and does not penetrate the mesophyll cells of treated leaves. We thus propose that P. aphidis acts via several mechanisms, and is an excellent candidate biocontrol agent.
Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Resistência à Doença/efeitos dos fármacos , Etilenos/farmacologia , Doenças das Plantas/microbiologia , Ustilaginales/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Botrytis/efeitos dos fármacos , Botrytis/fisiologia , Contagem de Colônia Microbiana , Controle Biológico de Vetores , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Ustilaginales/efeitos dos fármacos , Ustilaginales/crescimento & desenvolvimentoRESUMO
BACKGROUND: The yeast Metschnikowia fructicola is an antagonist with biological control activity against postharvest diseases of several fruits. We performed a transcriptome analysis, using RNA-Seq technology, to examine the response of M. fructicola with citrus fruit and with the postharvest pathogen, Penicillium digitatum. RESULTS: More than 26 million sequencing reads were assembled into 9,674 unigenes. Approximately 50% of the unigenes could be annotated based on homology matches in the NCBI database. Based on homology, sequences were annotated with a gene description, gene ontology (GO term), and clustered into functional groups. An analysis of differential expression when the yeast was interacting with the fruit vs. the pathogen revealed more than 250 genes with specific expression responses. In the antagonist-pathogen interaction, genes related to transmembrane, multidrug transport and to amino acid metabolism were induced. In the antagonist-fruit interaction, expression of genes involved in oxidative stress, iron homeostasis, zinc homeostasis, and lipid metabolism were induced. Patterns of gene expression in the two interactions were examined at the individual transcript level by quantitative real-time PCR analysis (RT-qPCR). CONCLUSION: This study provides new insight into the biology of the tritrophic interactions that occur in a biocontrol system such as the use of the yeast, M. fructicola for the control of green mold on citrus caused by P. digitatum.
Assuntos
Regulação Fúngica da Expressão Gênica , Metschnikowia/genética , Doenças das Plantas/genética , Citrus paradisi/química , Citrus paradisi/genética , Frutas/genética , Frutas/microbiologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Metschnikowia/metabolismo , Penicillium/genética , Penicillium/patogenicidade , Doenças das Plantas/microbiologiaRESUMO
Pseudozyma spp. are yeast-like fungi, classified in the Ustilaginales, which are mostly epiphytic or saprophytic and are not pathogenic to plants. Several Pseudozyma species have been reported to exhibit biological activity against powdery mildews. However, previous studies have reported that Pseudozyma aphidis, which can colonize plant surfaces, is not associated with the collapse of powdery mildew colonies. In this report, we describe a novel P. aphidis strain and study its interactions with its plant host and the plant pathogen Botrytis cinerea. This isolate was found to secrete extracellular metabolites that inhibit various fungal pathogens in vitro and significantly reduce B. cinerea infection in vivo. Moreover, P. aphidis sensitized Arabidopsis (Arabidopsis thaliana) plants' defense machinery via local and systemic induction of pathogenesis-related1 (PR1) and plant defensin1.2 (PDF1.2) expression. P. aphidis also reduced B. cinerea infection, locally and systemically, in Arabidopsis mutants impaired in jasmonic acid (JA) or salicylic acid (SA) signaling. Thus, in addition to direct inhibition, P. aphidis may inhibit B. cinerea infection via induced resistance in a manner independent of SA, JA, and Nonexpressor of PR1 (NPR1). P. aphidis primed the plant defense machinery and induced stronger activation of PDF1.2 after B. cinerea infection. Finally, P. aphidis fully or partially reconstituted PR1 and PDF1.2 expression in npr1-1 mutant and in plants with the SA hydroxylase NahG transgene, but not in a jasmonate resistant1-1 mutant, after B. cinerea infection, suggesting that P. aphidis can bypass the SA/NPR1, but not JA, pathway to activate PR genes. Thus, either partial gene activation is sufficient to induce resistance, or the resistance is not directed solely through PR1 and PDF1.2 but probably through other pathogen-resistance genes or pathways as well.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Basidiomycota/fisiologia , Ciclopentanos/metabolismo , Resistência à Doença/imunologia , Oxilipinas/metabolismo , Doenças das Plantas/imunologia , Ácido Salicílico/metabolismo , Arabidopsis/microbiologia , Basidiomycota/crescimento & desenvolvimento , Basidiomycota/isolamento & purificação , Basidiomycota/ultraestrutura , Botrytis/fisiologia , Botrytis/ultraestrutura , Solanum lycopersicum/microbiologia , Interações Microbianas , Mutação/genética , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Folhas de Planta/ultraestruturaRESUMO
The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.
Assuntos
Hemípteros/microbiologia , Rickettsia/isolamento & purificação , Estruturas Animais/microbiologia , Animais , Feminino , Trato Gastrointestinal/microbiologia , Masculino , Microscopia , Oócitos/microbiologia , Ovário/microbiologia , Testículo/microbiologiaRESUMO
BACKGROUND: Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium. FINDINGS: We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids. CONCLUSIONS: The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.
RESUMO
BACKGROUND: Botrytis cinerea is a haploid necrotrophic ascomycete which is responsible for 'grey mold' disease in more than 200 plant species. Broad molecular research has been conducted on this pathogen in recent years, resulting in the sequencing of two strains, which has generated a wealth of information toward developing additional tools for molecular transcriptome, proteome and secretome investigations. Nonetheless, transformation protocols have remained a significant bottleneck for this pathogen, hindering functional analysis research in many labs. RESULTS: In this study, we tested three different transformation methods for B. cinerea: electroporation, air-pressure-mediated and sclerotium-mediated transformation. We demonstrate successful transformation with three different DNA constructs using both air-pressure- and sclerotium-mediated transformation. CONCLUSIONS: These transformation methods, which are fast, simple and reproducible, can expedite functional gene analysis of B. cinerea.
Assuntos
Biotecnologia/métodos , Botrytis/genética , Técnicas de Transferência de Genes , Transformação Genética , DNA Fúngico/análise , Eletroporação , Hifas/genéticaRESUMO
Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as "maturases"), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns "hosts." These are all predicted to reside within mitochondria and may therefore act "in-trans" in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.