Hernan G. Rosli

A Draft Genome Sequence of Nicotiana benthamiana to Enhance Molecular Plant-Microbe Biology Research

Nicotiana benthamiana is a widely used model plant species for the study of fundamental questions in molecular plant-microbe interactions and other areas of plant biology. This popularity derives from its well-characterized susceptibility to diverse pathogens and, especially, its amenability to virus-induced gene silencing and transient protein expression methods. Here, we report the generation of a 63-fold coverage draft genome sequence of N. benthamiana and its availability on the Sol Genomics Network for both BLAST searches and for downloading to local servers. The estimated genome size of N. benthamiana is 3 Gb (gigabases). The current assembly consists of approximately 141,000 scaffolds, spanning 2.6 Gb with 50% of the genome sequence contained within scaffolds >89 kilobases. Of the approximately 16,000 N. benthamiana unigenes available in GenBank, >90% are represented in the assembly. The usefulness of the sequence was demonstrated by the retrieval of N. benthamiana orthologs for 24 immunity-associated genes from other species including Ago2, Ago7, Bak1, Bik1, Crt1, Fls2, Pto, Prf, Rar1, and mitogen-activated protein kinases. The sequence will also be useful for comparative genomics in the Solanaceae family as shown here by the discovery of microsynteny between N. benthamiana and tomato in the region encompassing the Pto and Prf genes.

iTAK: A Program for Genome-wide Prediction and Classification of Plant Transcription Factors, Transcriptional Regulators, and Protein Kinases.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity

BackgroundMicrobe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components.ResultsWe describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms.ConclusionsOur transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system.

β-Xylosidase in strawberry fruit: Isolation of a full-length gene and analysis of its expression and enzymatic activity in cultivars with contrasting firmness

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 β-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 β-xylosidases from higher plants. The recombinant protein obtained by over-expressing FaXyl1 in Escherichia coli had β-xylosidase activity against the artificial substrate p-nitrophenyl β-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 β-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 β-xylosidase activity in all ripening stages analyzed.

The SGN VIGS Tool: User-Friendly Software to Design Virus-Induced Gene Silencing (VIGS) Constructs for Functional Genomics

Virus-induced gene silencing (VIGS) is a fast and powerful method to study gene function in plants (Burch-Smith et al., 2004). It is based on plant defense mechanisms against viral gene replication and allows high-throughput silencing of genes of interest (Senthil-Kumar and Mysore, 2014). The molecular mechanisms involved in post-transcriptional gene silencing (PTGS) have been studied intensively, and its steps are well known. The silencing process begins with the recognition through Dicer-like ribonucleases (DCL) of double-stranded RNA (dsRNA) that is generated during viral replication.

α-L-arabinofuranosidase from strawberry fruit: cloning of three cDNAs, characterization of their expression and analysis of enzymatic activity in cultivars with contrasting firmness.

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.

Validation of reference transcripts in strawberry (Fragaria 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.

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

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.

Functional genomics of tomato for the study of plant immunity.

Tomato (Solanum lycopersicum), along with many other economically valuable species, belongs to the Solanaceae family. Understanding how plants in this family defend themselves against pathogens offers the opportunity of improving yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques that allow determination of changes in transcript abundance during pathogen attack. Such changes can implicate the affected gene as participating in plant defense. Testing the involvement of these candidate genes in defense has relied largely on posttranscriptional gene silencing, particularly virus-induced gene silencing. We discuss how functional genomics has played a key role in our current understanding of the defense response in tomato and related species and what are the challenges and opportunities for the future.