Identification of Transcripts Involved in Resistance Responses to Leaf Spot Disease Caused by Cercosporidium personatum in Peanut (Arachis hypogaea).

ABSTRACT Late leaf spot disease caused by Cercosporidium personatum is one of the most destructive foliar diseases of peanut (Arachis hypogaea) worldwide. The objective of this research was to identify resistance genes in response to leaf spot disease using microarray and real-time polymerase chain reaction (PCR). To identify transcripts involved in disease resistance, we studied the gene expression profiles in two peanut genotypes, resistant or susceptible to leaf spot disease, using cDNA microarray containing 384 unigenes selected from two expressed sequenced tag (EST) cDNA libraries challenged by abiotic and biotic stresses. A total of 112 spots representing 56 genes in several functional categories were detected as up-regulated genes (log(2) ratio > 1). Seventeen of the top 20 genes, each matching gene with known function in GenBank, were selected for validation of their expression levels using real-time PCR. The two peanut genotypes were also used to study the functional analysis of these genes and the possible link of these genes to the disease resistance trait. Microarray technology and real-time PCR were used for comparison of gene expression. The selected genes identified by microarray analysis were validated by real-time PCR. These genes were more greatly expressed in the resistant genotype as a result of response to the challenge of C. personatum than in the susceptible genotype. Further investigations are needed to characterize each of these genes in disease resistance. Gene probes could then be developed for application in breeding programs for marker-assisted selection.

Aphid biology: expressed genes from alate Toxoptera citricida, the brown citrus aphid.

The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. To better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing project of cDNA clones from alate aphids. Similar large-scale expressed sequence tag (EST) sequencing projects from other insects have provided a vehicle for answering biological questions relating to development and physiology. Although there is a growing database in GenBank of ESTs from insects, most are from Drosophila melanogaster and Anopheles gambiae, with relatively few specifically derived from aphids. However, important morphogenetic processes are exclusively associated with piercing-sucking insect development and sap feeding insect metabolism. In this paper, we describe the first public data set of ESTs from the brown citrus aphid, T. citricida. The cDNA library was derived from alate adults due to their significance in spreading viruses (e.g., citrus tristeza virus). Over 5180 cDNA clones were sequenced, resulting in 4263 high-quality ESTs. Contig alignment of these ESTs resulted in 2124 total assembled sequences, including both contiguous sequences and singlets. Approximately 33% of the ESTs currently have no significant match in either the non-redundant protein or nucleic acid databases. Sequences returning matches with an E-value of < or = -10 using BLASTX, BLASTN, or TBLASTX were annotated based on their putative molecular function and biological process using the Gene Ontology classification system. These data will aid research efforts in the identification of important genes within insects, specifically aphids and other sap feeding insects within the Order Hemiptera.

Electrophoretic and immunological evidence of unique proteins in leaves of citrus trees: application to citrus blight detection.

From the leaf tissue of healthy and blighted citrus trees 10-30 kDa soluble fractions were compared to find biochemical markers of tissue in the disease state. Using a non-denaturing extraction technique coupled with ultrafiltration, a resulting 10-30 kDa healthy and citrus blight fraction was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two distinct and adjacent bands for blight at an Mr of approximately 12,500 were separated. These bands were visible with Coomassie Brilliant Blue and silver stain but were negative to glycoprotein stains. An antiserum prepared against proteins isolated by preparative electrophoresis reacted only with the blight fractions and was distinctly different from healthy fractions when Western blotted. Only the gel region (Mr 12,500-13,000) of citrus blight sources was positive to the antiserum when compared with disease and nondisease stress sources. Results indicate that identification of specific proteins may be a way to diagnose the onset of citrus blight prior to visible tree symptoms.

Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck).

Flower buds of ;Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix (14)CO(2) into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total (14)C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the (14)C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO(2) per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO(2) into different compounds suggests that this CO(2) assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.

Allergenic cross-reactivity between Callistemon citrinis and Melaleuca quinquenervia pollens.

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH4B) and melaleuca (NH4M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH4B and NH4M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH4B1-NH4B4 and NH4M1-NH4M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH4B1 and NH4M1, respectively, contained allergenic components. These allergenic components, NH4B1 and NH4M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.

Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard.

Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with (13)C(6)[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned.