Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-<i>Pseudomonas pathosystem</i>

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.Instituto de Fisiología Vegeta

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-<i>Pseudomonas pathosystem</i>

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.Instituto de Fisiología Vegeta

Tomato Wall-Associated Kinase SlWak1 Depends on Fls2/Fls3 to Promote Apoplastic Immune Responses to <i>Pseudomonas syringae</i>

Wall-associated kinases (Waks) are important components of plant immunity against various pathogens, including the bacterium Pseudomonas syringae pv. tomato (Pst). However, the molecular mechanisms of their role(s) in plant immunity are largely unknown. In tomato (Solanum lycopersicum), wall-associated kinase 1 (SlWak1), has been implicated in pattern recognition receptor (PRR)-triggered immunity (PTI) because its transcript abundance increases significantly after treatment with the flagellin-derived, microbe-associated molecular patterns flg22 and flgII-28, which activate the PRRs Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Δwak1) using CRISPR/Cas9 gene editing technology and investigated the role of SlWak1 in tomato–Pst interactions. Late PTI responses activated in the apoplast by flg22 or flgII-28 were compromised in Δwak1 plants, but PTI at the leaf surface was unaffected. The Δwak1 plants developed fewer callose deposits than wild-type plants, but retained early PTI responses such as generation of reactive oxygen species and activation of mitogen-activated protein kinases upon exposure to flg22 and flgII-28. Induction of Wak1 gene expression by flg22 and flgII-28 was greatly reduced in a tomato mutant lacking Fls2 and Fls3, but induction of Fls3 gene expression by flgII-28 was unaffected in Δwak1 plants. After Pst inoculation, Δwak1 plants developed disease symptoms more slowly than Δfls2.1/2.2/3 mutant plants, although ultimately, both plants were similarly susceptible. SlWak1 coimmunoprecipitated with both Fls2 and Fls3, independently of flg22/flgII-28 or of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and is important at later stages of PTI in the apoplast.Los datos de secuencia de este artículo se pueden encontrar en Plant Genome Editing Database (http://plantcrispr.org) como Solyc09g014720Instituto de Fisiología Vegeta

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-<i>Pseudomonas pathosystem</i>

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.Instituto de Fisiología Vegeta

Efecto de la sobreexpresión de CBM-FaEXP2 en frutilla

Uno de los factores determinantes de la calidad y vida poscosecha de frutos carnosos es su firmeza, impuesta en gran medida por la resistencia mecánica que ofrece la pared celular vegetal. El fenómeno del ablandamiento de frutos es un proceso altamente regulado en el que participan diversos actores en forma concertada y redundante. Las expansinas (EXPs) son proteínas no hidrolíticas relevantes en procesos dónde es necesaria la relajación de la pared, tal como lo es el fenómeno de ablandamiento. Estas proteínas se caracterizan por presentar un módulo catalítico similar al de las glicosil-hidrolasas (carente de actividad hidrolítica) y un módulo de unión a carbohidratos (CBM) con fuerte afinidad por celulosa. En el presente trabajo se analizó el efecto de la sobreexpresión del CBM de la expansina dos de Fragaria x ananassa (FaExpa2) sobre el ablandamiento del fruto, usando Fragaria vesca cv. Hawaii 4 como sistema modelo, bajo la hipótesis de que la expresión de una proteína con la capacidad de unirse a carbohidratos, pero carente de actividad hidrolítica sobre los mismos genera una competencia por el sustrato con el resto de las enzimas involucradas en el catabolismo de la pared celular, provocando una reducción global de la degradación de la pared celular. Se analizaron distintos parámetros de calidad, la actividad expansina y poligalacturonasa en frutos transgénicos sobreexpresantes de CBM-FaEXPa2.Trabajo publicado en Castagnini, Juan Manuel; Luz Marina Zapata; Liliana Mabel Gerard (eds.). Libro de Trabajos Completos I Congreso Argentino de Biología y Tecnología Poscosecha. IX Jornadas Argentinas de Biología y Tecnología Poscosecha. Paraná: Universidad Nacional de Entre Ríos. UNER, 2018.Facultad de Ciencias Agrarias y Forestales (FCAF

Efecto de la sobreexpresión de CBM-FaEXP2 en frutilla

Uno de los factores determinantes de la calidad y vida poscosecha de frutos carnosos es su firmeza, impuesta en gran medida por la resistencia mecánica que ofrece la pared celular vegetal. El fenómeno del ablandamiento de frutos es un proceso altamente regulado en el que participan diversos actores en forma concertada y redundante. Las expansinas (EXPs) son proteínas no hidrolíticas relevantes en procesos dónde es necesaria la relajación de la pared, tal como lo es el fenómeno de ablandamiento. Estas proteínas se caracterizan por presentar un módulo catalítico similar al de las glicosil-hidrolasas (carente de actividad hidrolítica) y un módulo de unión a carbohidratos (CBM) con fuerte afinidad por celulosa. En el presente trabajo se analizó el efecto de la sobreexpresión del CBM de la expansina dos de Fragaria x ananassa (FaExpa2) sobre el ablandamiento del fruto, usando Fragaria vesca cv. Hawaii 4 como sistema modelo, bajo la hipótesis de que la expresión de una proteína con la capacidad de unirse a carbohidratos, pero carente de actividad hidrolítica sobre los mismos genera una competencia por el sustrato con el resto de las enzimas involucradas en el catabolismo de la pared celular, provocando una reducción global de la degradación de la pared celular. Se analizaron distintos parámetros de calidad, la actividad expansina y poligalacturonasa en frutos transgénicos sobreexpresantes de CBM-FaEXPa2.Trabajo publicado en Castagnini, Juan Manuel; Luz Marina Zapata; Liliana Mabel Gerard (eds.). Libro de Trabajos Completos I Congreso Argentino de Biología y Tecnología Poscosecha. IX Jornadas Argentinas de Biología y Tecnología Poscosecha. Paraná: Universidad Nacional de Entre Ríos. UNER, 2018.Facultad de Ciencias Agrarias y Forestales (FCAF

Efecto de la sobreexpresión de CBM-FaEXP2 en frutilla

Uno de los factores determinantes de la calidad y vida poscosecha de frutos carnosos es su firmeza, impuesta en gran medida por la resistencia mecánica que ofrece la pared celular vegetal. El fenómeno del ablandamiento de frutos es un proceso altamente regulado en el que participan diversos actores en forma concertada y redundante. Las expansinas (EXPs) son proteínas no hidrolíticas relevantes en procesos dónde es necesaria la relajación de la pared, tal como lo es el fenómeno de ablandamiento. Estas proteínas se caracterizan por presentar un módulo catalítico similar al de las glicosil-hidrolasas (carente de actividad hidrolítica) y un módulo de unión a carbohidratos (CBM) con fuerte afinidad por celulosa. En el presente trabajo se analizó el efecto de la sobreexpresión del CBM de la expansina dos de Fragaria x ananassa (FaExpa2) sobre el ablandamiento del fruto, usando Fragaria vesca cv. Hawaii 4 como sistema modelo, bajo la hipótesis de que la expresión de una proteína con la capacidad de unirse a carbohidratos, pero carente de actividad hidrolítica sobre los mismos genera una competencia por el sustrato con el resto de las enzimas involucradas en el catabolismo de la pared celular, provocando una reducción global de la degradación de la pared celular. Se analizaron distintos parámetros de calidad, la actividad expansina y poligalacturonasa en frutos transgénicos sobreexpresantes de CBM-FaEXPa2.Trabajo publicado en Castagnini, Juan Manuel; Luz Marina Zapata; Liliana Mabel Gerard (eds.). Libro de Trabajos Completos I Congreso Argentino de Biología y Tecnología Poscosecha. IX Jornadas Argentinas de Biología y Tecnología Poscosecha. Paraná: Universidad Nacional de Entre Ríos. UNER, 2018.Facultad de Ciencias Agrarias y Forestales (FCAF

Validation of reference transcripts in strawberry (<i>Fragaria</i> spp.)

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.Facultad de Ciencias ExactasInstituto de Fisiología Vegeta

Tomato wall-associated kinase SlWak1 acts in an Fls2- and Fls3-dependent manner to promote apoplastic immune responses to <i>Pseudomonas syringae</i>

Wall-associated kinases (Waks) are known to be important components of plant immunity against various pathogens including Pseudomonas syringae pv. tomato (Pst) although their molecular mechanisms are largely unknown. In tomato, SlWak1 has been implicated in immunity because its transcript abundance increases significantly in leaves after treatment with the flagellin-derived peptides flg22 and flgII-28, which activate the receptors Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Δwak1) using CRISPR/Cas9 and investigated the role of SlWak1 in tomato-Pst interactions. PTI activated in the apoplast by flg22 or flgII-28 was compromised in Δwak1 plants but PTI at the leaf surface was unaffected. The Δwak1 plants developed fewer callose deposits than wild-type plants but retained the ability to generate reactive oxygen species and activate MAPKs in response to flg22 and flgII-28. The induction of Wak1 gene expression by flg22 and flgII-28 was greatly reduced in a tomato mutant lacking Fls2 and Fls3 but induction of Fls3 gene expression by flgII-28 was unaffected in Δwak1 plants. After Pst inoculation, Δwak1 plants developed disease symptoms more slowly than Δfls2.1/fls2.2/fls3 mutant plants, although both plants ultimately were similarly susceptible. SlWak1 co-immunoprecipitated with both Fls2 and Fls3 independently of flg22/flgII-28 or Bak1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and plays an important role at later stages of the PTI in the apoplast.Instituto de Fisiología Vegeta

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

Plants mount defense responses by recognizing indications of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) contains two MAMPs, flg22 and flgII-28, that are recognized by tomato receptors Flagellin sensing 2 (Fls2) and Flagellin sensing 3 (Fls3), respectively. It is unknown to what degree each receptor contributes to immunity and if they promote immune responses using the same molecular mechanisms. Characterization of CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants revealed that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, striking differences were observed in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species (ROS) and an increase in transcript abundance of 44 tomato genes, with two genes serving as reporters for Fls3. Fls3 had greater in vitro kinase activity and interacted differently with the Pst effector AvrPtoB as compared to Fls2. Using chimeric Fls2/Fls3 proteins, we found that no receptor domain was solely responsible for the Fls3 sustained ROS, suggesting involvement of multiple structural features. This work reveals differences in the immunity outputs between Fls2 and Fls3, suggesting they use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.Instituto de Fisiología Vegeta