The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

A family of Arabidopsis plasma membrane receptors presenting animal beta-integrin domains.

A cDNA clone, AtELP1 (Arabidopsis thaliana EGF receptor-like protein) was isolated from an Arabidopsis cDNA library with an oligonucleotide probe corresponding to a highly conserved region of animal beta-integrins. The cloning of this cDNA was previously reported and it has been proposed that AtELP might be a receptor involved in intracellular trafficking. In the present work, using two specific independent sets of anti-peptide antibodies, we show that AtELP1 is mainly located in the plasma membrane, supporting another function for this protein. Structural studies, using methods for secondary structure prediction, indicated the presence of cysteine-rich domains specific to beta-integrins. Database searches revealed that AtELP1 is a member of a multigenic family composed of at least six members in A. thaliana. Northern blot analysis of AtELP1, 2b and 3 was performed on mRNA extracted from cells cultured in normal and stressed conditions, and from several organs and plants submitted to biotic or abiotic stresses. All the genes are expressed at different levels in the same conditions, but preferentially in roots, fruits and leaves in response to water deficit.

Osmotic stress activated expression of an Arabidopsis plasma membrane-associated protein: sequence and predicted secondary structure.

A cDNA clone At.MAMI (Arabidopsis thaliana membrane-associated mannitol-induced) was isolated from an Arabidopsis cDNA expression library by immunoselection. The cDNA was full-length (1.18 kb) with an open reading frame of 798 nucleotides encoding a 265 amino acid protein. The sequence of At.MAMI did not show any significant identity with other genes, as well as the deduced amino acid sequence with other proteins. However, prediction methods for the secondary structure of MAMI-30, together with homologous domains revealed some identity with VAP-33, a protein involved in membrane trafficking in neuronal tissues. In contrast to VAP-33, MAMI-30 did not exhibit a transmembrane domain, but positively charged loop regions could be involved in membrane anchoring. Indeed, MAMI-30 was immunodetected in purified plasma membrane from Arabidopsis cells. The gene was responsive to low turgor in Arabidopsis and its expression regulated developmentally. In addition, reduction of turgor caused a higher accumulation of mRNAs.