Robert E. Farrell

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

BackgroundThe necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.ResultscDNA were isolated from M.26 (susceptible) and G.41 (resistant) apple tissues collected 2 h and 48 h after challenge with a virulent E. amylovora strain or mock (buffer) inoculated. To identify differentially expressed transcripts, electrophoretic banding patterns were obtained from cDNAs. In the AFLP experiments, M.26 and G.41 showed different patterns of expression, including genes specifically induced, not induced, or repressed by E. amylovora. In total, 190 ESTs differentially expressed between M.26 and G.41 were identified using 42 pairs of AFLP primers. cDNA-AFLP analysis of global EST expression in a resistant and a susceptible apple genotype identified different major classes of genes. EST sequencing data showed that genes linked to resistance, encoding proteins involved in recognition, signaling, defense and apoptosis, were modulated by E. amylovora in its host plant. The expression time course of some of these ESTs selected via a bioinformatic analysis has been characterized.ConclusionThese data are being used to develop hypotheses of resistance or susceptibility mechanisms in Malus to E. amylovora and provide an initial categorization of genes possibly involved in recognition events, early signaling responses the subsequent development of resistance or susceptibility. These data also provided potential candidates for improving apple resistance to fire blight either by marker-assisted selection or genetic engineering.

Comparative expression and transcript initiation of three peach dehydrin genes

Dehydrin genes encode proteins with demonstrated cryoprotective and antifreeze activity, and they respond to a variety of abiotic stress conditions that have dehydration as a common component. Two dehydrins from peach (Prunus persica L. [Batsch.]) have been previously characterized; here, we describe the characterization of a third dehydrin from peach bark, PpDhn3, isolated by its response to low temperature. The expression of all three dehydrin genes was profiled by semi-quantitative reverse transcription PCR, and transcript initiation was mapped for all three genes using the RNA ligase-mediated 5′ rapid amplification of cDNA ends technique. PpDhn3 transcripts from bark collected in December or July, as well as transcripts from developing fruit, initiated at a single site. Although most of the PpDhn1 transcripts initiated at a similar position, those from young fruit initiated much further upstream of the consensus TATA box. Bark and fruit transcripts encoding PpDhn2 initiated ca. 30 bases downstream of a consensus TATA box; however, transcripts from ripe fruit initiated further upstream. Ripe fruit transcripts of PpDhn2 contain a 5′ leader intron which is predicted to add some 34 amino acids to the N-terminal methionine of the cognate protein when properly processed. Secondary structure prediction of sequences surrounding the TATA box suggests that conformational transitions associated with decreasing temperature contribute to the regulation of expression of the cold-responsive dehydrin genes. Taken together these results reveal new, unexpected levels of gene regulation contributing to the overall expression pattern of peach dehydrins.

Increase in antioxidant gene transcripts, stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure

A pretreatment of the yeast, Candida oleophila, with 5 mM H(2)O(2) for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H(2)O(2)), high temperature (40 °C), and low pH (pH 4). Compared with non-stress-adapted yeast cells, stress-adapted cells exhibited better control of apple fruit infections by Penicillium expansum and Botrytis cinerea and had initially higher growth rates in apple wounds. Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semiquantitative reverse transcription-PCR. Seven antioxidant genes were upregulated. The elevated expression of these genes was associated with less accumulation of reactive oxygen species and a lower level of protein and lipid oxidation under subsequent stresses. These data support the premise that induction of abiotic stress tolerance in biocontrol yeast can improve biocontrol efficacy by upregulation of genes involved in the amelioration of oxidative stress.

Multiple transcripts of a gene for a leucine-rich repeat receptor kinase from morning glory (Ipomoea nil) originate from different TATA boxes in a tissue-specific manner

TATA boxes are the most common regulatory elements found in the promoters of eukaryotic genes because they are associated with basal transcription initiation by RNA polymerase II. Often only a single TATA element is found in a given promoter, and tissue-, stage- and/or stimulus-specific expression occurs because the TATA box is associated with other cis -acting elements that enhance or repress transcription. We used software tools for gene analysis to assist in locating potential TATA box(es) in an AT-rich region of the promoter of a gene, inrpk1, which codes for a leucine-rich receptor protein kinase in morning glory (Ipomoea nil). Through the use of RT-PCR and various combinations of forward primers bracketing most of the promoter region we were able to define the 5′-ends of transcripts in this region. The region was then targeted for analysis by RNA Ligase-Mediated-5′ Rapid Amplification of cDNA Ends (RLM-5′ RACE) to identify the transcript initiation site(s). Positioning of initiation sites with respect to TATA boxes identified by gene analysis tools allowed us to identify three operational TATA elements which regulate basal transcription from this gene. Two TATA boxes were responsible for all of the inrpk1 transcripts found in leaves and cotyledons, and about 25–30% of the transcripts in roots. A third TATA box was involved only in expression in roots and accounted for the remaining 50–70% of root transcripts. RNAs expressed from this element lack two potentially functional upstream AUG codons, and may be translated more efficiently than transcripts originating from the other TATA boxes.

Genes responding to water deficit in apple ( Malus × domestica Borkh.) roots

BackgroundIndividual plants adapt to their immediate environment using a combination of biochemical, morphological and life cycle strategies. Because woody plants are long-lived perennials, they cannot rely on annual life cycle strategies alone to survive abiotic stresses. In this study we used suppression subtractive hybridization to identify genes both up- and down-regulated in roots during water deficit treatment and recovery. In addition we followed the expression of select genes in the roots, leaves, bark and xylem of ‘Royal Gala’ apple subjected to a simulated drought and subsequent recovery.ResultsIn agreement with studies from both herbaceous and woody plants, a number of common drought-responsive genes were identified, as well as a few not previously reported. Three genes were selected for more in depth analysis: a high affinity nitrate transporter (MdNRT2.4), a mitochondrial outer membrane translocase (MdTOM7.1), and a gene encoding an NPR1 homolog (MpNPR1-2). Quantitative expression of these genes in apple roots, bark and leaves was consistent with their roles in nutrition and defense.ConclusionsAdditional genes from apple roots responding to drought were identified using suppression subtraction hybridization compared to a previous EST analysis from the same organ. Genes up- and down-regulated during drought recovery in roots were also identified. Elevated levels of a high affinity nitrate transporter were found in roots suggesting that nitrogen uptake shifted from low affinity transport due to the predicted reduction in nitrate concentration in drought-treated roots. Suppression of a NPR1 gene in leaves of drought-treated apple trees may explain in part the increased disease susceptibility of trees subjected to dehydrative conditions.

Electrophoresis of RNA

Electrophoresis is a chromatography technique by which a mixture of charged molecules is separated according to size when placed in an electric field. The accurate determination of the size of RNA species is just as important as deduction of the molecular weight of any other macromolecules subjected to electrophoresis. This is accomplished by comparing the electrophoresis of an RNA sample against molecular weight standards. The successful electrophoresis of RNA is accomplished in two parts: RNA is denatured prior to loading the gel; and during the electrophoresis, conditions that support and maintain the denatured state are established. These steps will ensure that like species of RNA co-migrate, and the best possible resolution is achieved when gels are poured thinly and run at low voltage. The choice of denaturing system and gel matrix is determined primarily by the size range of the RNAs to be separated and the most commonly used RNA denaturants for agarose gel electrophoresis are formaldehyde and urea. Several gel staining techniques are used for the visualization of nucleic acids in agarose gels such as ethidium bromide, SYBR green, SYBR gold, SYBR safe, GelStar, silver staining, acridine orange, and methylene blue. The proper maintenance and use of electrophoresis gel boxes and power supplies is of utmost importance for the safety of all.

Analysis of Nuclear RNA

This chapter presents a comparison of the traditional Northern analysis, nuclease protection assays, nuclear run-off assay, and the polymerase chain reaction. Two basic approaches are used to study the mechanism of transcription of specific genes. In one approach, the rate of transcription is measured in intact nuclei by the incorporation of labeled precursor nucleotides into RNA transcripts. But the other approach involves the isolation of steady-state nuclear RNA by Northern analysis or nuclease protection assay. The RNA polymerase will “run-off” the end of the template, producing transcripts of uniform length as soon as the conditions supporting transcriptional activation are met. The quantity of RNA produced is therefore an indicator of the efficiency of transcription. The degree of success of the nuclear run-off assay is entirely dependent on the speed with which nuclei can be isolated from intact cells and radiolabeled. The relative rate of transcription of several genomic sequences can be evaluated simultaneously by the nuclear run-off assay. The nuclear run-off assay facilitates the assessment of the transcription rate of individual genes by the elongation of nascent polyribonucleotides in the presence of labeled NTP precursor. In addition, steady-state nuclear RNA, as well as labeled RNA produced by nuclear run-off, is essential for understanding the processing of the primary transcript. Northern analysis of nuclear RNA yields quantitative data based on signal intensity of discrete bands, but also provides a qualitative component that cannot be discerned by Polymerase chain reaction or nuclease protection.

Multiple Transcript Initiation as a Mechanism for Regulating Gene Expression

Transcription is the intermediary process that copies a DNA-encoded gene into a form which is either functional in its own right (stable RNAs, such as ribosomal or transfer RNAs) or can be decoded by the translational machinery into a functional protein. Transcripts destined for translation are called messenger RNAs (mRNAs), since they act as go-betweens from DNA to protein. Although RNAs are transcribed as single-stranded molecules, most can assume complicated secondary and tertiary structures that are critical for proper functioning. As a result, each mRNA contains not only the sequence information required to synthesize a protein, but also structural components that can regulate mRNA localization, stability, and translation efficiency. Thus the initiation of transcription occupies a preeminent place in the regulation of gene expression.

RNA Isolation Strategies

This chapter focuses on the process of isolation of ribonucleic acid (RNA) from biological sources enriched in ribonucleases (RNase) and the protocols for the analysis of steady-state transcripts. Steady-state RNA is the one that is isolated from the nucleus or cytoplasm by direct cell lysis. RNA is chemically and biologically more labile at elevated temperatures (65°) and in the presence of alkali. These intrinsic handling difficulties are further compounded by aggressive activities of a variety of resilient RNase. Protocols for the isolation of RNA begin with cellular lysis mediated by buffers that typically fall into one of two categories depending on the degree of cellular disruption and concomitant RNase inhibition: (1) those consisting of harsh chaotropic agents such as one of the guanidinium salts, sodium dodecyl sulfate (SDS), N-laurylsarcosine (sarcosyl), urea, phenol, or chloroform, which disrupt the plasma membrane and subcellular organelles and which simultaneously inactivate RNase; and (2) those that gently solubilize the plasma membrane while maintaining nuclear and other organelle integrity, such as the non-ionic, hypotonic lysis buffers. The disadvantage of using gentle lysis buffers is that the lysis buffer alone is not sufficiently chaotropic to fully inhibit RNase activity; and the drawback of harsh chaotropic procedures is that the investigator is not able to discriminate between cytoplasmic and nuclear RNA, as there is no method for separating hnRNA from spliced messenger RNA (mRNA) once these two populations have been mixed.

The complete peach dehydrin family: characterization of three recently recognized genes

Three genes encoding dehydrins have been previously described from peach. In the course of searching the peach genome, three additional members of this stress-associated family were recognized, PpDhn4-6. PpDhn1 and 6 have no introns, whereas the remaining four genes have a single intron located near the 3′ end of the serine (S) tract. PpDHN2 was the only dehydrin with a predicted basic pI; pI predictions for the other dehydrins ranged from about 5.3 to about 6.3. None of the peach dehydrins have tryptophan residues, but, in contrast to most dehydrins, three (PpDHN1, 3, and 4) have one or more cysteine residues. Phylogenetic analysis indicated a close relationship between PpDhn1 and 6 and PpDhn3 and 4. Expression analysis under low temperature and dehydration confirmed that PpDhn2 is the major responder to drought, while both PpDhn1 and 6 respond exclusively to cold. Comparison of the first 500 base pairs upstream of the translation start site revealed the presence of cis-elements associated with low temperature and drought/osmotic/salt and hormone response regulation.