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%.

Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew.

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.

Transcriptional down-regulation by abscisic acid of pathogenesis-related beta-1,3-glucanase genes in tobacco cell cultures.

Class I isoforms of beta-1,3-glucanases (betaGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense. Tobacco (Nicotiana tabacum L.) betaGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation. Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin. We report a novel pattern of betaGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants. Abscisic acid (ABA) at a concentration of 10 micron markedly inhibited the induction of betaGLU I but not of CHN I. RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of betaGLU and CHN are induced in cell culture and that ABA inhibits steady-state betaGLU I mRNA accumulation. Comparable inhibition of beta-glucuronidase expression by ABA was observed for cells transformed with a tobacco betaGLU I gene promoter/beta-glucuronidase reporter gene fusion. Taken together, the results strongly suggest that ABA down-regulates transcription of betaGLU I genes. This raises the possibility that some of the ABA effects on plant-defense responses might involve betaGLU I.

Pathogenesis-related functions of plant beta-1,3-glucanases investigated by antisense transformation--a review.

Plant beta-1,3-glucanases (beta Glu) have been implicated in several physiological and developmental processes, e.g., cell division, microsporogenesis, pollen germination, fertilization and seed germination. These enzymes, particularly the antifungal class-I vacuolar isoforms, are also believed to be part of the defences of plants against fungal infection. The function of beta Glu in tobacco and Nicotiana sylvestris has been investigated by antisense transformation. Transformation with GLA, the gene encoding the A isoform of tobacco class-I beta Glu, in reverse orientation regulated by the strong cauliflower mosaic virus 35S RNA promoter effectively and specifically blocked the induction of class-I beta Glu. This induction was in response to ethylene treatment and following infection with the pathogenic fungus, Cercospora nicotianae, tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV). Nevertheless, the plants compensated for this deficiency by producing a functionally equivalent (i.e., "ersatz') enzyme or enzymes. The fact that compensation occurred specifically in response to infection suggests that beta Glu activity has an important role in pathogenesis. Antisense transformation substantially reduced lesion size and number in virus-infected local-lesion hosts. These results suggest novel antisense-based strategies for protecting plants against virus infection. They also raise the intriguing possibility that viruses use a defence mechanism of the host, production of antifungal beta Glu, to promote their own replication and spread.

Decreased Susceptibility to Viral Disease of [beta]-1,3-Glucanase-Deficient Plants Generated by Antisense Transformation.

Antifungal class I [beta]-1,3-glucanases are believed to be part of the constitutive and induced defenses of plants against fungal infection. Unexpectedly, mutants deficient in these enzymes generated by antisense transformation showed markedly reduced lesion size, lesion number, and virus yield in the local-lesion response of Havana 425 tobacco to tobacco mosaic virus (TMV) and of Nicotiana sylvestris to tobacco necrosis virus. These mutants also showed decreased severity of mosaic disease symptoms, delayed spread of symptoms, and reduced yield of virus in the susceptible response of N. sylvestris to TMV. The symptoms of disease in the responses of both plant species were positively correlated with [beta]-1,3-glucanase content in a series of independent transformants. Taken together, these results provide direct evidence that [beta]-1,3-glucanases function in viral pathogenesis. Callose, a substrate for [beta]-1,3-glucanase, acts as a physical barrier to the spread of virus. Callose deposition in and surrounding TMV-induced lesions was increased in the [beta]-1,3-glucanase-deficient, local-lesion Havana 425 host, suggesting as a working hypothesis that decreased susceptibility to virus resulted from increased deposition of callose in response to infection. Our results suggest novel means, based on antisense transformation with host genes, for protecting plants against viral infection. These observations also raise the intriguing possibility that viruses can use a defense response of the host against fungal infection[mdash]production of [beta]-1,3-glucanases[mdash]to promote their own replication and spread.

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

Physiological compensation in antisense transformants: specific induction of an "ersatz" glucan endo-1,3-beta-glucosidase in plants infected with necrotizing viruses.

Plant class I glucan endo-1,3-beta-glucosidases (beta-1,3-glucanase; 1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) have been implicated in development and defense against pathogen attack. Nevertheless, beta-1,3-glucanase deficiencies generated by antisense transformation of Nicotiana sylvestris and tobacco have little biological effect. We report here that another beta-1,3-glucanase activity is induced in these deficient mutants after infection with necrotizing viruses. Induction of class I beta-1,3-glucanase was markedly inhibited in leaves of N. sylvestris and tobacco antisense transformants infected with tobacco necrosis virus and tobacco mosaic virus, respectively. A serologically distinct beta-1,3-glucanase activity was present in the infected antisense transformants but was absent in both healthy and infected control plants and in antisense transformants treated with the stress hormone ethylene. Immunoblot analyses, localization studies, and measurements of antibody specificity indicate that this compensatory beta-1,3-glucanase activity is an intracellular enzyme different from known tobacco beta-1,3-glucanases. Therefore, plants can compensate for a deficiency in enzyme activity by producing a functionally equivalent replacement--i.e., "ersatz"--protein or proteins. The fact that compensation for beta-1,3-glucanase activity occurs in response to infection argues strongly for an important role of these enzymes in pathogenesis.

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots.

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl(2) and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [(3)H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.