Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

Background  

The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.     

Sensitivity of different E. coli and Salmonella strains in mutagenicity testing calculated on the basis of selected literature

Two microbial screening test systems for gene (point) mutations, the Salmonella typhimurium assay (TA1535, TA1537, TA1538, TA98 and TA100) and the Escherichia coli WP2 reverse-mutation system (WP2, WP2uvrA, WP2pKM101 and WP2uvrApKM101), were compared with regard to sensitivity toward a broad spectrum of compounds that cause base-pair or frameshift mutations and that have known carcinogenic qualities. Based on available published literature we found that all 44 carcinogens and 9 non-carcinogens examined in both test systems also met with criteria for data acceptance drawn up by us. The results obtained are: firstly, that the Salmonella assay is decidedly better validated than the E. coli WP2 test; and secondly, that the E. coli test system sensitivity (91%) is fully on a par with the sensitivity of the Salmonella assay (72%). This last is in divergence from earlier reports, e.g. Brusick et al. (1980), and this difference must be ascribed to the new plasmid-containing strains. The many compounds not tested in the E. coli department result in fewer false negatives in the E. coli test system and their omission constitutes a bias in favour of the E. coli assay. By eliminating compounds that are negative in Salmonella and dropped from the WP2 analysis owing to insufficient data, the sensitivity of the Salmonella system is raised to 84% as compared with 91% for the WP2 assay. The results further indicate that some of the tester strains are superfluous, and show an exceedingly sensitive test can be performed by combining the best tester strains from the two test systems.     

Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize

Sugarcane mosaic virus (SCMV) is an important virus pathogen both in European and Chinese maize production, causing serious losses in grain and forage yield in susceptible cultivars. Two major resistance loci confer resistance to SCMV, one located on chromosome 3 (Scmv2) and one on chromosome 6 (Scmv1). We developed a large isogenic mapping population segregating in the Scmv2, but not the Scmv1 region, to minimize genetic variation potentially affecting expression of SCMV resistance. We fine mapped Scmv2 to a region of 0.28 cM, covering a physical distance of 1.3426 Mb, and developed six new polymorphic SSR markers based on publicly available BAC sequences within this region. At present, we still have three recombinants left between Scmv2 and the nearest polymorphic marker on either side of the Scmv2 locus. The region showed synteny to a 1.6 Mb long sequence on chromosome 12 in rice. Analysis of the public B73 BAC library as well as the syntenic rice region did not reveal any similarity to known resistance genes. However, four new candidate genes with a possible involvement in movement of virus were detected.     

Immunohistochemical localisation of ubiquitin and the proteasome in sunflower (Helianthus annuus cv. Giganteus)

This study provides an immunohistochemical demonstration of the involvement of the ubiquitin- and proteasome-dependent pathway during differentiation and organogenesis in plants. The localisation of ubiquitin and the proteasome was studied in meristems, leaves, stems and roots of sunflower (Helianthus annuus L. cv. Giganteus). By using a new technique that enhances very low antigen signals, we obtained information on the structural distribution of the ubiquitin- and proteasome-dependent pathway, and of the importance of this pathway during organogenesis and plant development. Ubiquitin and the proteasome showed overall similarities in their cellular localisation. The highest antigenic signal was observed in the root and shoot apical meristems, in leaf primordia and vascular tissue. The cambium showed less expression than the apical meristems. During adventitious root formation in cuttings, no sign of increased expression was observed within dedifferentiating tissue, but as organogenesis progressed, the antigenic signal of ubiquitin and the proteasome gradually increased in the developing roots. Comparison of immunochemical results and Western blots demonstrated that important changes in the cellular antigen signal could only be detected by immunochemistry.     

The Pic19 NBS-LRR gene family members are closely linked to Scmv1, but not involved in maize resistance to sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) is the causal pathogen for a severe mosaic virus disease of maize worldwide. In our previous research, the maize resistance gene analog (RGA) Pic19 and its three cognate BAC contigs were mapped to the same region as the SCMV resistance gene Scmv1. Here we report the isolation and characterization of the Pic19R gene family members from the inbred line FAP1360A, which shows complete resistance to SCMV. Two primer pairs were designed based on the conserved regions among the known Pic19 paralogs and used for rapid amplification of cDNA ends of FAP1360A. Six full-length cDNAs, corresponding to the Pic19R-1 to -6 paralogs, were obtained. Three of them (Pic19R-1 to -3) had uninterrupted coding sequences and were, therefore, regarded as candidates for the Scmv1 gene. A total of 18 positive BAC clones harboring the Pic19R-2 to -5 paralogs were obtained from the FAP1360A BAC library and assembled into two BAC contigs. Two markers, tagging Pic19R-2 and -3 and Pic19R-4, were developed and used to genotype a high-resolution mapping population segregating solely for the Scmv1 locus. Although closely linked, none of these three Pic19R paralogs co-segregated with the Scmv1 locus. Analysis of the Pic19R family indicated that the Pic19R-1 paralog is identical to the known Rxo1 gene conferring resistance to rice bacterial streak disease and none of the other Pic19R paralogs seems to be involved in resistance to SCMV.     

Response of young barley plants to CO2 enrichment

Barley (Hordeum vulgare L. cv. Digger) was grown for 22 d inenclosed chambers with a CO₂ enrichment of 35, 155, 400 or 675µmol CO₂ mol1. CO₂ enrichment increased photosyntheticcapacity in the plants grown at either of the two highest levelsof pCO₂. A CO₂ enrichment of 675µmol CO₂ caused a significantincrement of shoot dry weight, whereas no changes were observedin fresh weight, chlorophyll or protein levels. At a light intensityof 860µmol m^–2s^–1 CO₂ enrichment caused photosyntheticcapacity to increase by 250%, whereas no effect was observedat 80 µmol m^–2 s^–1. Over time, photosynthesisdecreased by 70% independent of CO₂. A time-dependent increasein the level of extractable fructose was observed whereas totalextractable carbohydrate only changed slightly. Key words: Carbohydrates, CO₂ enrichment, Hordeum vulgare, photosynthesis, respiration     

Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.

The gene action of 2 sugarcane mosaic virus (SCMV) resistance loci in maize, Scmv1 and Scmv2, was evaluated for potyvirus resistance in an isogenic background. All 4 homozygous and 5 heterozygous isogenic genotypes were produced for introgressions of the resistant donor (FAP1360A) alleles at both loci into the susceptible parent (F7) genetic background using simple sequence repeat markers. For SCMV and maize dwarf mosaic virus (MDMV), virus symptoms appeared rapidly in the 3 homozygous genotypes, with susceptibility alleles fixed at 1 or both loci. Although the 9 isogenic genotypes revealed a high level of resistance to Zea mosaic virus (ZeMV), the same 3 homozygous genotypes were only partially resistant. This indicates that 1 resistance gene alone is not sufficient for complete resistance against SCMV, MDMV, and ZeMV. Scmv1 showed strong early and complete dominant gene action to SCMV, but it gradually became partially dominant. Scmv2 was not detected at the beginning, showing dominant gene action initially and additive gene action at later stages. Both genes interacted epistatically (for a high level of resistance, at least 1 resistance allele at each of both loci is required). This implies that double heterozygotes at the 2 loci are promising for producing SCMVresistant hybrids. Results are discussed with respect to prospects for isolation of SCMV and MDMV resistance genes.