RESUMO
Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ascomicetos/fisiologia , Quitina/análogos & derivados , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Proteínas Serina-Treonina Quinases/metabolismo , Vitis/enzimologia , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Quitina/metabolismo , Quitina/farmacologia , Quitosana , Oligossacarídeos , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Vitis/genética , Vitis/imunologiaRESUMO
Stomata remain abnormally opened and unresponsive to abscisic acid in grapevine leaves infected by downy mildew. This deregulation occurs from 3 days postinoculation and increases concomitantly with leaf colonization by the pathogen. Using epidermal peels, we demonstrated that the active compound involved in this deregulation is located in the apoplast. Biochemical assays showed that the active compound present in the apoplastic fluids isolated from Plasmopara viticola-infected grapevine leaves (IAF) is a CysCys bridge-independent, thermostable and glycosylated protein. Fractionation guided assays based on chromatography coupled to stomatal response and proteomic analysis allowed the identification of both plant and pathogen proteins in the active fraction obtained from IAF. Further in silico analysis and discriminant filtrations based on the comparison between predictions and experimental indications lead to the identification of two Vitis vinifera proteins as candidates for the observed stomatal deregulation.
Assuntos
Glicoproteínas/metabolismo , Oomicetos/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Estômatos de Plantas/metabolismo , Vitis/metabolismo , Sequência de Aminoácidos , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/microbiologia , Cromatografia por Troca Iônica , Simulação por Computador , Proteínas Fúngicas/metabolismo , Glicoproteínas/classificação , Glicoproteínas/genética , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Oomicetos/fisiologia , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Estômatos de Plantas/genética , Estômatos de Plantas/microbiologia , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Vitis/genética , Vitis/microbiologiaRESUMO
Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.
Assuntos
Botrytis/genética , Resistência à Doença/genética , Frutas/genética , Doenças das Plantas/genética , Vitis/genética , Botrytis/patogenicidade , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/microbiologia , Ciclopentanos/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/microbiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Interações Hospedeiro-Patógeno/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/metabolismo , Sesquiterpenos/metabolismo , Estilbenos/metabolismo , Virulência/genética , Vitis/crescimento & desenvolvimento , Vitis/microbiologia , FitoalexinasRESUMO
⢠The role of flagellin perception in the context of plant beneficial bacteria still remains unclear. Here, we characterized the flagellin sensing system flg22-FLAGELLIN SENSING 2 (FLS2) in grapevine, and analyzed the flagellin perception in the interaction with the endophytic plant growth-promoting rhizobacterium (PGPR) Burkholderia phytofirmans. ⢠The functionality of the grapevine FLS2 receptor, VvFLS2, was demonstrated by complementation assays in the Arabidopsis thaliana fls2 mutant, which restored flg22-induced H2O2 production and growth inhibition. Using synthetic flg22 peptides from different bacterial origins, we compared recognition specificities between VvFLS2 and AtFLS2. ⢠In grapevine, flg22-triggered immune responses are conserved and led to partial resistance against Botrytis cinerea. Unlike flg22 peptides derived from Pseudomonas aeruginosa or Xanthomonas campestris, flg22 peptide derived from B. phytofirmans triggered only a small oxidative burst, weak and transient defense gene induction and no growth inhibition in grapevine. Although, in Arabidopsis, all the flg22 epitopes exhibited similar biological activities, the expression of VvFLS2 into the fls2 background conferred differential flg22 responses characteristic for grapevine. ⢠These results demonstrate that VvFLS2 differentially recognizes flg22 from different bacteria, and suggest that flagellin from the beneficial PGPR B. phytofirmans has evolved to evade this grapevine immune recognition system.
Assuntos
Burkholderia/fisiologia , Endófitos/crescimento & desenvolvimento , Epitopos/imunologia , Flagelina/imunologia , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Vitis/imunologia , Vitis/microbiologia , Sequência de Aminoácidos , Arabidopsis/fisiologia , Botrytis/efeitos dos fármacos , Botrytis/fisiologia , Burkholderia/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Contagem de Colônia Microbiana , Simulação por Computador , Resistência à Doença/efeitos dos fármacos , Endófitos/efeitos dos fármacos , Flagelina/farmacologia , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Proteínas de Plantas/química , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/química , Especificidade da Espécie , Vitis/efeitos dos fármacos , Vitis/crescimento & desenvolvimentoRESUMO
BACKGROUND: For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. 'Chardonnay'). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy. RESULTS: By comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. 'Chardonnay'. These results are directly correlated with Cas9 expression level. CONCLUSIONS: Taken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.
RESUMO
The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.
RESUMO
Black rot (Guignardia bidwellii) and downy mildew (Plasmopara viticola) are two major grapevine diseases against which the development of efficient biocontrol solutions is required in a context of sustainable viticulture. This study aimed at evaluating and comparing the efficacy and modes of action of bacterial culture supernatants from Bacillus velezensis Buz14 and B. ginsengihumi S38. Both biocontrol agents (BCA) were previously demonstrated as highly effective against Botrytis cinerea in grapevines. In semi-controlled conditions, both supernatants provided significant protection against black rot and downy mildew. They exhibited antibiosis against the pathogens by significantly decreasing G. bidwellii mycelial growth, but also the release and motility of P. viticola zoospores. They also significantly induced grapevine defences, as stilbene production. The LB medium, used for the bacterial cultures, also showed partial effects against both pathogens and induced plant defences. This is discussed in terms of choice of experimental controls when studying the biological activity of BCA supernatants. Thus, we identified two bacterial culture supernatants as new potential biocontrol products exhibiting multi-spectrum antagonist activity against different grapevine key pathogens and having a dual mode of action.
RESUMO
The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.
Assuntos
Nicotiana , Imunidade Vegetal , Proteínas de Plantas , Plantas Geneticamente Modificadas , Nicotiana/genética , Nicotiana/virologia , Nicotiana/imunologia , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Doenças das Plantas/virologia , Doenças das Plantas/imunologia , Ácido Salicílico/metabolismo , Regulação da Expressão Gênica de Plantas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Vírus do Mosaico do Tabaco/fisiologia , Phytophthora/fisiologia , Phytophthora/patogenicidadeRESUMO
The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.
RESUMO
Using plant defense elicitors to protect crops against diseases is an attractive strategy to reduce chemical pesticide use. However, development of elicitors remains limited because of variable effectiveness in the field. In contrast to fungicides that directly target pathogens, elicitors activate plant immunity, which depends on plant physiological status. Other products, the biostimulants, can improve certain functions of plants. In this study, the objective was to determine whether a biostimulant via effects on grapevine physiology could increase effectiveness of a defense elicitor. A new methodology was developed to study biostimulant activity under controlled conditions using in vitro plantlets. Both biostimulant and defense elicitor used in the study were plant extracts. When added to the culture medium, the biostimulant accelerated the beginning of plantlet growth and affected the shoot and root development. It also modified metabolomes and phytohormone contents of leaves, stems, and roots. When applied on shoots, the defense elicitor changed metabolite and phytohormone contents, but effects were different depending on whether plantlets were biostimulated or controls. Defense responses and protection against Plasmopara viticola (downy mildew agent) were induced only for plantlets previously treated with the biostimulant, Therefore, the biostimulant may act by priming the defense elicitor action. In this study, a new method to screen biostimulants active on grapevine vegetative growth was used to demonstrate that a biostimulant can optimize the efficiency of a plant defense elicitor.
RESUMO
The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.
Assuntos
Enzimas/metabolismo , Oomicetos/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Amido/metabolismo , Vitis/fisiologia , Metabolismo dos Carboidratos , Clorofila/metabolismo , Enzimas/genética , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Hexoses/análise , Hexoses/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oomicetos/patogenicidade , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Polissacarídeos/análise , Polissacarídeos/metabolismo , RNA de Plantas/genética , Amido/análise , Trealose/metabolismo , Vitis/enzimologia , Vitis/genética , Vitis/microbiologia , alfa-Amilases/genética , alfa-Amilases/metabolismo , beta-Amilase/genética , beta-Amilase/metabolismoRESUMO
The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic pterostilbene, which plays a crucial role in grapevine interaction with Plasmopara viticola and Botrytis cinerea. As a resveratrol O-methyltransferase (ROMT) gene involved in pterostilbene synthesis was recently identified, we investigated the accumulation of the corresponding transcripts to those of two other stilbene biosynthesis related genes phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) in response to pathogen infection. Using three computer-based statistical methods and C(t) values or LRE method generated values as input data, we have first identified two reference genes (VATP16 and 60SRP) suitable for normalization of qPCR expression data obtained in grapevine leaves and berries infected by P. viticola and B. cinerea, respectively. Next, we have highlighted that the expression of ROMT is induced in P. viticola-infected leaves and also in B. cinerea-infected berries, confirming the involvement of pterostilbene in grapevine defenses.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Estilbenos/metabolismo , Vitis/genética , Primers do DNA/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Software , Estatística como Assunto , Vitis/enzimologiaRESUMO
The root system plays an essential role in the development and physiology of the plant, as well as in its response to various stresses. However, it is often insufficiently studied, mainly because it is difficult to visualize. For grapevine, a plant of major economic interest, there is a growing need to study the root system, in particular to assess its resistance to biotic and abiotic stresses, understand the decline that may affect it, and identify new ecofriendly production systems. In this context, we have evaluated and compared three distinct growing methods (hydroponics, plane, and cylindric rhizotrons) in order to describe relevant architectural root traits of grapevine cuttings (mode of grapevine propagation), and also two 2D- (hydroponics and rhizotron) and one 3D- (neutron tomography) imaging techniques for visualization and quantification of roots. We observed that hydroponics tubes are a system easy to implement but do not allow the direct quantification of root traits over time, conversely to 2D imaging in rhizotron. We demonstrated that neutron tomography is relevant to quantify the root volume. We have also produced a new automated analysis method of digital photographs, adapted for identifying adventitious roots as a feature of root architecture in rhizotrons. This method integrates image segmentation, skeletonization, detection of adventitious root skeleton, and adventitious root reconstruction. Although this study was targeted to grapevine, most of the results obtained could be extended to other plants propagated by cuttings. Image analysis methods could also be adapted to characterization of the root system from seedlings.
RESUMO
Both plants and animals are endowed with sophisticated innate immune systems to combat microbial attack. In these multicellular eukaryotes, innate immunity implies the presence of cell surface receptors and intracellular receptors able to detect danger signal referred as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Membrane-associated pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are employed by these organisms for sensing different invasion patterns before triggering antimicrobial defenses that can be associated with a form of regulated cell death. Intracellularly, animals nucleotide-binding and oligomerization domain (NOD)-like receptors or plants nucleotide-binding domain (NBD)-containing leucine rich repeats (NLRs) immune receptors likely detect effectors injected into the host cell by the pathogen to hijack the immune signaling cascade. Interestingly, during the co-evolution between the hosts and their invaders, key cross-kingdom cell death-signaling macromolecular NLR-complexes have been selected, such as the inflammasome in mammals and the recently discovered resistosome in plants. In both cases, a regulated cell death located at the site of infection constitutes a very effective mean for blocking the pathogen spread and protecting the whole organism from invasion. This review aims to describe the immune mechanisms in animals and plants, mainly focusing on cell death signaling pathways, in order to highlight recent advances that could be used on one side or the other to identify the missing signaling elements between the perception of the invasion pattern by immune receptors, the induction of defenses or the transmission of danger signals to other cells. Although knowledge of plant immunity is less advanced, these organisms have certain advantages allowing easier identification of signaling events, regulators and executors of cell death, which could then be exploited directly for crop protection purposes or by analogy for medical research.
Assuntos
Morte Celular/imunologia , Células Vegetais/imunologia , Imunidade Vegetal/imunologia , Plantas/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Animais , Humanos , Imunidade Inata/imunologia , Inflamassomos/imunologia , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
Stomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H(2)O(2) production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA and elicitors share common signaling events, it led us to question whether stomatal movements and H(2)O(2) production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine-Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production.
Assuntos
Oomicetos/fisiologia , Estômatos de Plantas/fisiologia , Vitis/microbiologia , Ácido Abscísico/farmacologia , Peróxido de Hidrogênio/metabolismo , Imunidade Inata , Luz , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Estômatos de Plantas/microbiologia , Estômatos de Plantas/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Vitis/efeitos da radiaçãoRESUMO
Laminaran, a ß-(1â3)-glucan extracted from Laminaria digitata, is a known elicitor of plant defenses, but provides only low level of disease control in vineyard trials. In this context, laminaran was partly hydrophobized by grafting from 1.6 to 7.6 lauryl chains to the native saccharidic chain and the impact of sulfation of the hydrophobized glucans was studied. The activity of the different synthetized laminaran derivatives as antimicrobial agents against Plasmopara viticola, the causal agent of grape downy mildew, and as elicitors of defense reactions in planta, was evaluated. Our results showed that acylation imparts an antimicrobial activity to laminaran which is related to the degree of acylation, AL3, with 7.6 lauryl chains, being the most effective derivative. Sulfation of the acylated laminarans did not further increase the antimicrobial activity. Our results also demonstrated that the efficacy of AL3 against Plasmopara viticola was most likely due to the direct antimicrobial activity of the lauryl chains rather than to an elicitation of plant defenses.
Assuntos
Resistência à Doença , Glucanos/farmacologia , Oomicetos/metabolismo , Doenças das Plantas/microbiologia , Vitis , Anti-Infecciosos/farmacologia , Laminaria/metabolismo , Vitis/metabolismo , Vitis/microbiologiaRESUMO
Crude extracts of Vitis vinifera canes represent a natural source of stilbene compounds with well characterized antifungals properties. In our trials, exogenous application of a stilbene extract (SE) obtained from grape canes on grapevine leaves reduces the necrotic lesions caused by Botrytis cinerea. The SE showed to possess a direct antifungal activity by inhibiting the mycelium growth. The activation of some grapevine defense mechanism was also investigated. H2O2 production and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascades as well as accumulation of stilbenoid phytoalexins were explored on grapevine cell suspension. Moreover, the transcription of genes encoding for proteins affecting defense responses was analyzed on grapevine plants. The SE induced some grapevine defense mechanisms including MAPK activation, and the expression of pathogenesis-related (PR) genes and of a gene encoding the glutathione-S-transferase 1 ( GST1) . By contrast, treatment of grapevine leaves with SE negatively regulates de novo stilbene production.
Assuntos
Botrytis/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Doenças das Plantas/microbiologia , Extratos Vegetais/farmacologia , Caules de Planta/química , Vitis/química , Vitis/microbiologia , Botrytis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estilbenos/farmacologiaRESUMO
Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.
Assuntos
Bacillus subtilis/fisiologia , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Doenças das Plantas/microbiologia , Vitis/imunologia , Vitis/microbiologia , Bacillus subtilis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucanos/biossíntese , Peronospora/efeitos dos fármacos , Peronospora/fisiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Vitis/efeitos dos fármacos , Vitis/genéticaRESUMO
In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.