RESUMO

MAIN CONCLUSION: CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.

Assuntos

Solanum lycopersicum , Fosfolipases Tipo C , Fosfolipases Tipo C/metabolismo , Solanum lycopersicum/genética , Sistemas CRISPR-Cas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxilipinas/metabolismo , Melhoramento Vegetal , Botrytis/metabolismo , Fosfolipases/genética , Fosfolipases/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas

RESUMO

Plants defend themselves against pathogens using a two-layered immune system. The first response, pattern-triggered immunity (PTI), is activated upon recognition of microbe-associated molecular patterns (MAMPs). Virulent bacteria such as Pseudomonas syringae pv. tomato (Pst), deliver effector proteins into the plant cell to promote susceptibility. However, some plants possess resistance (R) proteins that recognize specific effectors leading to the activation of the second response, effector-triggered immunity (ETI). Resistant tomatoes such as Río Grande-PtoR recognize two Pst effectors (AvrPto and AvrPtoB) through the host Pto/Prf complex and activate ETI. We previously showed that the transcription factors (TF) WRKY22 and WRKY25 are positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens in Nicotiana benthamiana. Here, the CRISPR-Cas9 technique was used to develop three knockout tomato lines for either one or both TFs. The single and double mutants were all compromised in Pto/Prf-mediated ETI and had a weaker PTI response. The stomata apertures in all of the mutant lines did not respond to darkness or challenge with Pst DC3000. The WRKY22 and WRKY25 proteins both localize in the nucleus, but we found no evidence of a physical interaction between them. The WRKY22 TF was found to be involved in the transcriptional regulation of WRKY25, supporting the idea that they are not functionally redundant. Together, our results indicate that both WRKY TFs play a role in modulating stomata and are positive regulators of plant immunity in tomato.

Assuntos

Solanum lycopersicum , Solanum lycopersicum/genética , Pseudomonas syringae/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/metabolismo , Mutação , Imunidade Vegetal/genética , Doenças das Plantas/microbiologia

RESUMO

To succeed in plant invasion, phytopathogenic bacteria rely on virulence mechanisms to subvert plant immunity and create favorable conditions for growth. This process requires a precise regulation in the production of important proteins and metabolites. Among them, the family of compounds known as polyamines have attracted considerable attention as they are involved in important cellular processes, but it is not known yet how phytopathogenic bacteria regulate polyamine homeostasis in the plant environment. In the present study, we performed a meta-analysis of publicly available transcriptomic data from experiments conducted on bacteria to begin delving into this topic and better understand the regulation of polyamine metabolism and its links to pathogenicity. We focused our research on Pseudomonas syringae, an important phytopathogen that causes disease in many economically valuable plant species. Our analysis discovered that polyamine synthesis, as well as general gene expression activation and energy production are induced in the early stages of the disease. On the contrary, synthesis of these compounds is inhibited whereas its transport is upregulated later in the process, which correlates with the induction of virulence genes and the metabolism of nitrogen and carboxylic acids. We also found that activation of plant defense mechanisms affects bacterial polyamine synthesis to some extent, which could reduce bacterial cell fitness in the plant environment. Furthermore, data suggest that a proper bacterial response to oxidative conditions requires a decrease in polyamine production. The implications of these findings are discussed.

RESUMO

KEY MESSAGE: NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors. Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.

Assuntos

Nicotiana/genética , Nicotiana/metabolismo , Imunidade Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Apoptose , Arabidopsis/genética , Proteínas de Arabidopsis , Morte Celular , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas/genética , Solanum lycopersicum/genética , Filogenia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/classificação , Pseudomonas syringae/patogenicidade , Fatores de Transcrição/classificação

RESUMO

Plants rely on (in)direct detection of bacterial pathogens through plasma membrane-localized and intracellular receptor proteins. Surface pattern-recognition receptors (PRRs) participate in the detection of microbe-associated molecular patterns (MAMPs) and are required for the activation of pattern-triggered immunity (PTI). Pathogenic bacteria, such as Pseudomonas syringae pv. tomato (Pst) deploys ~ 30 effector proteins into the plant cell that contribute to pathogenicity. Resistant plants are capable of detecting the presence or activity of effectors and mount another response termed effector-triggered immunity (ETI). In order to investigate the involvement of tomato's long non-coding RNAs (lncRNAs) in the immune response against Pst, we used RNA-seq data to predict and characterize those that are transcriptionally active in leaves challenged with a large set of treatments. Our prediction strategy was validated by sequence comparison with tomato lncRNAs described in previous works and by an alternative approach (RT-qPCR). Early PTI (30 min), late PTI (6 h) and ETI (6 h) differentially expressed (DE) lncRNAs were identified and used to perform a co-expression analysis including neighboring (± 100 kb) DE protein-coding genes. Some of the described networks could represent key regulatory mechanisms of photosynthesis, PRR abundance at the cell surface and mitigation of oxidative stress, associated to tomato-Pst pathosystem.

Assuntos

Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , RNA Longo não Codificante , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Biologia Computacional/métodos , Ontologia Genética , Genoma de Planta , Estudo de Associação Genômica Ampla , Genômica/métodos , Interações Hospedeiro-Patógeno/imunologia , Solanum lycopersicum/imunologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia

RESUMO

BACKGROUND: Softening is one of the main features that determine fruit quality during strawberry (Fragaria x ananassa, Duch.) ripening and storage. Being closely related to textural changes, the molecular and biochemical bases underlying strawberry cell-wall metabolism is a matter of interest. Here we investigated the abundance of transcripts encoding putative strawberry endo-xylanases in plant tissues, during fruit ripening and under postharvest and hormonal treatments. Total xylanase activity and expression of related genes in strawberry varieties with contrasting firmness were analyzed. RESULTS: FaXynA and FaXynC mRNA abundance was significantly higher than FaXynB in each plant tissue studied. Higher total xylanase activity was detected at the end of the ripening of the softer cultivar ('Toyonoka') in comparison with the firmer one ('Camarosa'), correlating with the abundance of FaXynA and FaXynC transcripts. Postharvest 1-methylcyclopropene treatment up-regulated FaXynA and FaXynC expressions. FaXynC mRNA abundance decreased with heat treatment but the opposite was observed for FaXynA. Calcium chloride treatment down-regulated FaXynA and FaXynC expression. Both genes responded differently to plant growth regulators' exposure. FaXynC expression was down-regulated by auxins and gibberellins treatment and up-regulated by abscisic acid. FaXynA was up-regulated by auxins, while no changes in mRNA levels were evident by abscisic acid and gibberellins treatment. Ethephon exposure did not change FaXynA and FaXynC expressions. CONCLUSION: New knowledge about the presence of xylanases in ripening strawberry fruit and their response to postharvest and hormonal treatments is provided. Our findings suggest a role for endo-xylanases in hemicelluloses depolymerization and possibly in strawberry fruit softening. © 2020 Society of Chemical Industry.

Assuntos

Endo-1,4-beta-Xilanases/genética , Fragaria/genética , Frutas/enzimologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Ácido Abscísico/farmacologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Fragaria/química , Fragaria/efeitos dos fármacos , Fragaria/enzimologia , Frutas/química , Frutas/efeitos dos fármacos , Frutas/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Ácidos Indolacéticos/farmacologia , Cinética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo

RESUMO

RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.

Assuntos

Nicotiana/genética , Nicotiana/microbiologia , Proteínas de Plantas/genética , Pseudomonas fluorescens , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Algoritmos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Folhas de Planta/genética , Folhas de Planta/microbiologia , Pseudomonas fluorescens/patogenicidade

RESUMO

The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

Assuntos

Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Pseudomonas syringae , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Transcriptoma , Algoritmos , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Reprodutibilidade dos Testes , Análise de Sequência de RNA

RESUMO

Softening of fleshy fruits during ripening is associated to catabolism of cell wall components. In strawberry, pectin degradation, as well as loss of neutral sugars (mainly arabinose), increases during ripening, and probably contributes to fruit softening. In this work, we report the activity of alpha-l-arabinofuranosidase (alpha-l-arafase) and the expression of related genes in strawberry. Activity of alpha-l-arafase was measured during ripening of cultivars with contrasting firmness. An important increment in the specific activity of alpha-l-arafase was detected during ripening in both cultivars. However, in the softest one (Toyonoka) the specific activities were higher than in the firmest (Camarosa). A combination of semi quantitative reverse transcriptase-PCR (RT-PCR) with degenerate primers and a screening of a cDNA library allowed the isolation and cloning of three cDNAs encoding putative alpha-l-arafases (FaAra1, FaAra2 and FaAra3). The deduced proteins revealed that FaAras belong to the glycoside hydrolase family 51 and not to glycoside hydrolase family 3. Expression studies, carried out by means of Northern-blot and semi quantitative RT-PCR, revealed that FaAras were predominantly expressed in fruit tissue and detected over the entire ripening process. Due to similarity of FaAras sequences, Northern-blot analysis probably grouped the expression of the three genes. The expression was high at small green stage, decreased at white stage and increased thereafter. The increment of the expression from white to 50% red stage was more evident in the softest cultivar (Toyonoka). Semi quantitative RT-PCR analysis allowed determining the expression of individual FaAras. The expression of the three genes was detected in all developmental and ripening stages. However, differences in expression levels could be detected between cultivars. In the softest cultivar, the expression of the three FaAras was higher at 50% and 75% red stages, and in the case of FaAra3 a higher expression was found also at 100% red stage. Overall, specific activity of alpha-l-arafase was higher in the softest cultivar; such activity reflects the expression of at least three putative FaAra genes.

Assuntos

DNA Complementar/genética , Fragaria/enzimologia , Frutas/enzimologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Northern Blotting , Clonagem Molecular , Fragaria/genética , Fragaria/metabolismo , Frutas/genética , Frutas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos

RESUMO

Strawberry is a non-climateric fleshy fruit, which softens quickly and has short post-harvest life. Ripening is associated with an increment of pectin solubility and a reduction of the content of hemicelluloses. In this work, we have cloned the full-length cDNA encoding a ß-xylosidase (FaXyl1) from Fragaria×ananassa and we have characterized its expression in two strawberry cultivars with contrasting fruit firmness. The analysis of the predicted protein showed that FaXyl1 is closely related to other ß-xylosidases from higher plants. The recombinant protein obtained by over-expressing FaXyl1 in Escherichia coli had ß-xylosidase activity against the artificial substrate p-nitrophenyl ß-d-xilopyranoside. Differently from other bifunctional xylosidases, no α-l-arabinofuranosidase activity was detected in the recombinant enzyme. The expression of FaXyl1 gene was analyzed by northern-blot in Camarosa and Toyonaka strawberry cultivars, and compared with the corresponding protein data obtained by Western-blot and with the ß-xylosidase activity during ripening. The softest cultivar (Toyonaka) showed an early accumulation of FaXyl1 transcript and a higher expression of the corresponding protein during ripening, which correlates with a higher ß-xylosidase activity in all ripening stages analyzed.