A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5' end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR setup. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluoresceinlabeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT-PCR analysis is relatively flexible, and it could be as short as 56bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT-PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT-PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.

A complete set of candidate disease resistance (R) genes encoding nucleotide-binding sites (NBSs) was identified in the genome sequence of japonica rice (Oryza sativa L. var. Nipponbare). These putative R genes were characterized with respect to structural diversity, phylogenetic relationships and chromosomal distribution, and compared with those in Arabidopsis thaliana. We found 535 NBS-coding sequences, including 480 non-TIR (Toll/IL-1 receptor) NBS-LRR (Leucine Rich Repeat) genes. TIR NBS-LRR genes, which are common in A. thaliana, have not been identified in the rice genome. The number of non-TIR NBS-LRR genes in rice is 8.7 times higher than that in A. thaliana, and they account for about 1% of all of predicted ORFs in the rice genome. Some 76% of the NBS genes were located in 44 gene clusters or in 57 tandem arrays, and 16 apparent gene duplications were detected in these regions. Phylogenetic analyses based both NBS and N-terminal regions classified the genes into about 200 groups, but no deep clades were detected, in contrast to the two distinct clusters found in A. thaliana. The structural and genetic diversity that exists among NBS-LRR proteins in rice is remarkable, and suggests that diversifying selection has played an important role in the evolution of R genes in this agronomically important species.

The Vat resistance gene (in Cucumis melo L.) inhibits the transmission of non-persistent viruses by Aphis gossypii Glover, but does not affect transmission by Myzus persicae (Sulz.). To seewhether this difference was behaviourally determined, we investigated the stylet penetration behaviour of these two aphid species by recording EPGs (Electrical Penetration Graphs) of 8 and 20min on two sets of susceptible and resistant isogenic melon lines. During the 20min EPG study, inoculation with CMV (Cucumber Mosaic Virus) was also investigated. For both sets of isogenic lines, the two aphid species were able to detect the presence of Vat. The mean duration of individual intracellular punctures on the resistant genotypes was significantly reduced for both M. persicae and A. gossypii (-10% and -8% respectively for duration of pattern 'pd' in the 20min experiment); this reaction appeared faster for M. persicae, a species for which melon was not a suitable host-plant. Therefore, in contrast to Vat's antitransmission effect, this behavioural effect was not aphid species-specific. Also, the frequency of intracellular punctures on the resistant genotypes was significantly reduced in A. gossypii (but not in M. persicae): on average, this frequency dropped from ≈0.65pd·min-1 on the susceptible genotypes to ≈0.5pd·min-1 on the resistant ones. It was concluded that (intracellular?) chemical cues were detected very early by aphids probing on the resistant genotypes carrying the Vat gene. However, a comprehensive analysis of the behavioural traits of both aphids on the two genotypes could not alone account for the complete inhibition of transmission which was found only to occur for A. gossypii on resistant genotypes. None of the differences detected (between aphid species or between plant genotypes) could account for the Vat phenotype, although they may explain quantitative differences in ransmission efficiencies between aphid species. It was thus concluded that Vat effect was primarily chemically mediated. Finally, some intracellular punctures bearing typical subphases have been identified in both aphid species and were designated as 'long potential drops' (pd-L). For A. gossypii, these were observed early after plant contact and their mean duration was twice that of standard intracellular punctures (≈8.5s vs ≈4.2s). Although not necessary for CMV inoculation, the duration of such phases was positively correlated with a high transmission efficiency by A. gossypii on the susceptible genotype. The nature of this pattern and a putative mechanism of action of the Vat gene are discussed."

In themelon, the Vat (monogenic, dominant) resistance gene governs both an antixenotic reaction to the melon aphid Aphis gossypii Glover (Homoptera, Aphididae) and a resistance to non-persistent virus transmission, restricted to this vector species. We investigated the behavioural features and tissue localisation of the antixenosis resistance by the electrical penetration graph technique (EPG, DC system).We also compared the chemical composition in amino compounds and proteins of the phloem sap collected from two isogenic lines of melon (Cucumis melo L.), carrying the Vat gene or not. All behavioural and chemical data indicated that this resistance is onstitutive. EPG analysis clearly showed that access to phloem, although delayed by alterations in pathway activities, was not impaired in terms of frequency of access or initiation of feeding. The most striking feature was, however, a very reduced duration of ingestion from phloem of resistant plants, making this compartment one of the tissues where the effects of the Vat gene are unambiguously expressed. This was confirmed by clear differential activity of phloem extracts in artificial no-choice bioassays. Chemical analyses have shown that phloem saps from the two isogenic lines were extremely similar in profiles of ninhydrin positive compounds, and contained a low total amount of free amino acids (less than 10mM). Out of more than 40 distinguishable peaks in the chromatograms (protein and non-protein amino acids, as well as small peptides), only five differentiated the two genotypes. Two of them were increased in the resistant genotype: glutamic acid and a major unknown peak, probably a non-protein amino acid (different from pyrazolyl-alanine, a Cucumis-specific amino acid). The three others were depressed in resistant plants, and included the sulphur amino acid cystine and a peptide peak partly composed of the cysteine-ontaining peptide glutathione (reduced form). Sap collection also showed that phloem exudation rates, as well as total protein and glutathione levels, were depressed in phloem sap from resistant plants. Such data are all indicative of a modified phloem-sealing physiology, linked to sulfhydryl oxidation processes, in plants carrying the Vat gene. The originality of the mechanism of Vat resistance to aphids is discussed.