L. M. Kawchuk

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Tobacco Mosaic Virus Infection Results in an Increase in Recombination Frequency and Resistance to Viral, Bacterial, and Fungal Pathogens in the Progeny of Infected Tobacco Plants

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.

Genetic diversity of potato determined by random amplified polymorphic DNA analysis.

SummaryThe RAPD procedure was used to establish genetic diversity of 28 potato genotypes including siblings and genotypes with no immediate relationship. In addition amplified DNA from three parents and Solanum chacoense were compared with that from six progeny to determine the genetic relationships. Amplification of genomic DNA from the 28 genotypes using PCR and 12 decamer primers yielded 158 amplified DNA fragments, ranging in size from 490 to 3200 bp. A total of 128 unique RAPD fragments were observed among the 28 potato genotypes. Similarity measures and principal coordinate analysis generally reflected the expected trends in relationships of the full and half-sib potato genotypes. However there were important exceptions to this general trend and it appears that related varieties can be as genetically different as varieties with no immediate relationship. The data suggest that RAPD analysis used in conjunction with pedigree information can provide a superior measure of genetic divergence than analysis based solely on pedigree information.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plants RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0(PE), in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(PE) destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

Identification of a gene conferring high levels of resistance to Verticillium wilt in Solanum chacoense

Verticillium wilt (Verticillium albo-atrum) is an important disease affecting potato tuber yield and quality. In North America the major commercial cultivars are susceptible and management strategies for control of the pathogen rely mainly on soil fumigation and crop rotation. In this study 398 genotypes from accessions of Solanum berthaultii, S. chacoense, and S. tarijense were screened for resistance to Verticillium albo-atrum. Resistant genotypes were identified in all but two accessions; however, results indicate that tolerance is more common than resistance. We identified two genotypes in S. chacoense (PI 472819) that had low stem-colonization levels and also did not develop wilt symptoms when inoculated with V. albo-atrum. These genotypes and a susceptible genotype from PI 472810 (S. chacoense) were studied to determine genetic inheritance. Segregation ratios in F1, F2, and backcross populations indicated that resistance in one of the resistant genotypes (18-21R) was controlled by a single dominant gene. Transfer of the Vc gene to tetraploid germ plasm could provide effective and economical control of Verticillium wilt.

Characterization ofSolanum tuberosum simple sequence repeats and application to potato culiwar identification

With the continued introduction of new potato cultivars, accurate identification is becoming difficult but is essential for maintaining cultivar integrity and Plant Breeders’ Rights. Hypervariable DNA sequences, referred to as simple sequence repeats (SSRs) or microsatellites, have been reported to be an excellent source of genetic markers. To determine the abundance, distribution, and composition of SSRs withinSolanium tuberosum, 252 sequences were searched for tetranucleotide and smaller SSRs with a minimum length of 20 nucleotides and a maximum discrepancy of two nucleotides. In total, 40 unique SSRs were observed in the 252S. tuberosum sequences examined and occurred at a frequency of one SSR every 8.1 kb. To assess the ability of site-specific amplified SSRs to identify potato cultivars, a simple (TCAC)m and compound (TCAC)m • (CTT)n SSR 5’ to the starch synthase gene and a compound (C)p • (CT)q • (AT)r • (G)s SSR 5’ to the sequence encoding mature proteinase inhibitor I, were examined and shown to produce unique DNA profiles for 73 of 95 tetraploid cultivars. In total, 24 alleles were observed at these loci and the accurately sized amplified DNA products can be used to establish a database for cultivar identification. Site-specific amplified alleles were somatically stable and have been conserved in clonal variants of Russet Burbank independently maintained for almost seven decades, a characteristic essential for cultivar identification. As genetic markers, the abundant, informative, and easily examined site-specific amplified alleles of SSRs are ideal for quickly and accurately determining cultivar identity of S.tuberosum ssp.tuberosum.

Resistance to thiabendazole and thiophanate-methyl in canadian isolates ofFusarium sambucinum andHelminthosporium solani

Resistance to both thiabendazole (TBZ) and thiophanate-methyl (TPM) was observed in Canadian isolates ofFusarium sambucinum andHelminthosporium solani recovered from diseased potato tubers. Resistance was not found in isolates ofFusarium avenaceum, Fusarium culmorum, Fusarium equiseti andFusarium solani. A high level of resistance to both TBZ (EC50 180 to ≫500 mg/l) and TPM (EC50 ≫500 mg/l) was observed forH. solani isolates, whereasF. sambucinum exhibited a relatively low level of resistance to TBZ (EC50 34 to 71 mg/l) and a high level of resistance to TPM (growth uninhibited by 2,500 mg/l). The incidence ofF. sambucinum resistant to the benzimidazoles was lower (60%) than that observed forH. solani (95%)CompendioEn Canadá, se observó resistencia tanto al Tiabendazole como al MetilTiofanato, en aislamientos de Fusarium sambucinum y Helminthosporium solani obtenidos de tubérculos de papa enfermos. No se encontró resistencia en aislamientos de Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti y Fusarium solani. Se observó un alto nivel de resistencia tanto al TBZ (EC50 180 a ≫500 mg/dm3) como al TPM (EC50 ≫500 mg/dm3) para los aislamientos de H. solani, mientras que F. sambucinum mostró un nivel relativamente bajo de resistencia al TBZ (EC50 34 a 71 mg/dm3) y un alto nivel de resistencia al TPM (su crecimiento no fue inhibido por 2 500 mg/dm3). La incidencia de F. sambucinum resistente a los bencimidazoles fue menor (60%) que la observada para H. solani (95%)

Identification of potato cultivars and clonal variants by random amplified polymorphic DNA analysis

Random Amplified Polymorphic DNAs (RAPDs) were used to distinguish commercial potato cultivars and clonal variants of cultivars. Primer 131, one of four primers used, distinguished 30 of the 36 cultivars tested. All 36 commercial cultivars were distinguished using only two primers (131 and 184). The RAPD pattern of 20 unidentified potato cultivars was compared with known patterns of 36 cultivars. Each one of the 20 cultivars was correctly identified. Particular primers appear to produce greater numbers of both amplified DNA fragments and polymorphisms, and are therefore suited to RAPD identification of potato cultivars. Polymorphism was obtained between Russet Burbank Idaho D and Russet Burbank White Skin with primer 251 and between Viking and Purple Viking with primer 380. However, polymorphism was not observed between Norgold, Norland, Sebago and Superior clones using only 20 primers. The RAPD technique is much more likely to detect polymorphism, regardless of tissue or environmental factors, than isozyme analysis and is easier, less costly and faster than the RFLP procedure. Thus, RAPD analysis represents a highly useful method of distinguishing and identifying potato cultivars and clonal variants of cultivars.CompendioSe utilizaron DNAs Polimórficos Amplificados al Azar (RAPDs) para identificar cultivares comerciales de papa y variantes clonales de cultivares. El Imprimador 131, uno de cuatro imprimadores utilizados, identificó 30 de los 36 cultivares estudiados. Todos los 36 cultivares comerciales fueron identificados utilizando solamente dos imprimadores (131 y 184). El modelo RAPD de 20 cultivares no identificados fue comparado con modelos conocidos de 36 cultivares. Cada uno de los 20 cultivares fue correctamente identificado. Imprimadores específicos parecen producir números mayores tanto de fragmentos como de polimorfismos amplificados de DNA y son por lo tanto apropiados para la identificatión de cultivares de papa utilizando el RAPD. Se obtuvo polimorfismo entre Russet Burbank Idaho D y Russet Burbank White Skin con el imprimador 251 y entre Viking y Purple Viking con el imprimador 380. Sin embargo, utilizando solamente 20 imprimadores, no se observaron polimorfismos entre los clones de Norgold, Norland, Sebago y Superior. La técnica RAPD es más apropiada para detectar polismorfismos, independientemente del tejido o de los factores del ambiente, que el análisis de isoenzimas y es más fácil, menos costosa y más rápida que el procedimiento RFLP (polimorfismo de restricción de la longitud de los fragmentes). Por lo tanto, el análisis RAPD representa un método muy útil para distinguir e identificar cultivares de papa y variantes clonales de los mismos.

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5′-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome.

Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum

The entomopathogenic fungus Metarhizium anisopliae var. acridum is registered as a mycopesticide for acridid control in Africa and Australia. Traditionally, identification of M. anisopliae var. acridum infection in grasshoppers and locusts has relied upon development of fungal growth in infected cadavers. Conventional methods of detection of this entomopathogen in the environment and non-target organisms have been based on culture and bioassay. A PCR-based method for the detection of M. anisopliae var. acridum was developed. Sequence data from the distinct ITS rDNA regions facilitated the design of PCR primers that were used in PCR-based diagnostic assays for the detection of fungal DNA. The amplified sequence was 420 bp in length and specific to M. anisopliae var. acridum. Isolates of M. anisopliae var. anisopliae and M. flavoviride produced no PCR product with these primers. Other fungal entomopathogens, plant pathogens, mycopathogens, and soil saprophytes were also not detected by the pathogen-specific primers. The assay was also effective for the detection of M. anisopliae var. acridum DNA in the presence of soil DNA extracts and in infected grasshoppers.