Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.     

Biodiversity of marine nematodes in Australian sandy beaches from tropical and temperate regions

The universal occurrence and abundance of nematodes provides opportunities to investigate ecological factors that may influence biodiversity. Clarke and Warwick (2001) have proposed diversity indices average taxonomic distance(AvTD), variation in taxonomic distinctness (VarTD) for computing marine nematode biodiversity based on classification trees. Faith [Biological Conservation 61 (1992) 1] had previously proposed a diversity index based on phylogenetic trees, though not applied to nematodes. Clarke and Warwick (2001) also considered an index AvPD analogous to AvTD. These indices have been applied to five very large collections of free-living nematodes from three exposed sandy beaches in Australia. Two were from a beach close to Darwin in northern Australia, two from the temperate southeast coast of Australia and one from the south of the Australian mainland. The collections extend over a considerable range of latitude, from 12°26S to 38°33S and the controversial hypothesis that latitudinal gradients per se influence the biodiversity of marine nematode assemblages is examined. AvTD did not vary among collections and its value for any collection was indistinguishable from that of random samples of the same size drawn from the total species pool. VarTD showed no variation for three of the collections, but was slightly higher than expected for the mid-latitude beach, attributed to unevenness in the classification trees. AvTD and VarTD were not greatly affected by differences in sampling intensity. PD varied directly with the number of species found but observed PD did not differ from the PD of random samples of the same number of species taken from the total species pool. Thus, the observed variation in PD is wholly accounted for by variations in species richness. The number of species found increased with decreasing latitude. It appears that species richness by itself is an adequate index of biodiversity for the free-living nematodes of these sandy beaches, and more complex indices such as AvTD, VarTD and PD are unnecessary.     

A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.     

The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato

The necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broad-spectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved:S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPT- and S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT- and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein. Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.     

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid populations growth. Artificial diets were used to provide active (1, 10, 100 and 500 g ml^–1) and inactive (500 g ml^–1) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day populations doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.     

Mutan produced in potato amyloplasts adheres to starch granules

Production of water-insoluble mutan polymers in Kardal potato tubers was investigated after expression of a full-length (GtfI) and a truncated mutansucrase gene referred to as GtfICAT (GtfI without glucan-binding domain) from Streptococcus downei. Subsequent effects on starch biosynthesis at the molecular and biochemical levels were studied. Expression of the GtfICAT gene resulted in the adhesion of mutan material on starch granules, which stained red with erythrosine, and which was hydrolysed by exo-mutanase. In addition, GtfICAT-expressing plants exhibited a severely altered tuber phenotype and starch granule morphology in comparison to those expressing the full-length GtfI gene. In spite of that, no structural changes at the starch level were observed. Expression levels of the sucrose-regulated, AGPase and GBSSI genes were down-regulated in only the GTFICAT transformants, showing that GtfICAT expression interfered with the starch biosynthetic pathway. In accordance with the down-regulated AGPase gene, a lower starch content was observed in the GTFICAT transformants. Finally, the rheological properties of the GTFICAT starches were modified; they showed a higher retrogradation during cooling of the starch paste.     

Metabolic response of potato plants to an antisense reduction of the P-protein of glycine decarboxylase

Potato (Solanum tuberosum L. cv. Desiré) plants with reduced amounts of P-protein, one of the subunits of glycine decarboxylase (GDC), have been generated by introduction of an antisense transgene. Two transgenic lines, containing about 60–70% less P-protein in the leaves compared to wild-type potato, were analysed in more detail. The reduction in P-protein amount led to a decrease in the ability of leaf mitochondria to decarboxylate glycine. Photosynthetic and growth rates were reduced but the plants were viable under ambient air and produced tubers. Glycine concentrations within the leaves were elevated up to about 100-fold during illumination. Effects on other amino acids and on sucrose and hexoses were minor. Nearly all of the glycine accumulated during the day was metabolised during the following night. The data suggest that the GDC operates far below substrate saturation under normal conditions thus allowing a flexible and fast response to changes in the environment. Received: 4 March 2000 / Accepted: 26 July 2000     

Evolutionary relationships of trichostrongyloid nematodes (Strongylida) inferred from ribosomal DNA sequence data

The evolutionary relationships of 21 species of trichostrongyloid nematodes were determined by use of sequence data of the second internal transcribed spacer of the ribosomal DNA aligned according to secondary structure information. Irrespective of the method of analysis used, the topologies of the phylogenetic trees derived from the molecular data differed with respect to all four hypotheses proposed previously for the evolutionary relationships of the different subfamilies within the Trichostrongylidae based on morphological data. Thus, the molecular data set did not resolve the conflict between the four previous proposals for the subfamilial relationships. Nonetheless, all trees derived from the molecular data showed strong support for the exclusion of the genera Filarinema and Amidostomum from the clade containing the species within the family Trichostrongylidae. This represents a major difference from the most recent proposal of the systematics of the Trichostrongyloidea in which these two genera were included within the Trichostrongylidae. Therefore, the molecular data support an earlier systematic framework in which Filarinema and Amidostomum were considered to be sister groups of the Trichostrongyloidea.     

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.     

Insecticidal activity of avidin and streptavidin against four species of pest Lepidoptera

Two biotin-binding proteins, avidin and streptavidin, were found to be insecticidal to the larvae of four species of Lepidoptera – light brown apple moth, Epiphyas postvittana (Walker) (fam. Tortricidae), green-headed leaf-roller, Planotortrix octo (Dugdale) (fam. Tortricidae), brown-headed leaf-roller, Ctenopseustis obliquana (Walker) (fam. Tortricidae) and potato tuber moth, Phthorimaea operculella (Zeller) (fam. Gelechiidae). Mortality occurred in all species, but there was a wide range in susceptibility. P. operculella larvae were the most susceptible with an LC₅₀ of respectively, 2.3 g ml^–1 for avidin and 1.4 g ml^–1 for streptavidin after 9 days. E. postvittana larvae had an LC₅₀ of 43.4 g ml^–1 for avidin after 21 days, and C. obliquana larvae of 45.7 g ml^–1 for avidin after 28 days. Although significant mortality occurred in P.octo at the highest doses of avidin, there was no sufficient dose-mortality response to calculate an LC₅₀ for this species. For all species mortality curves were steep over a close range of doses followed by a plateau where mortality did not increase significantly with dose and did not reach 100%. Mortality was significantly affected by the amount of biotin in the diet on which the parental generation had been reared. Where this was rich in biotin, significant mortality of the offspring was much lower: larval offspring of a colony of E. postvittana, reared for five generations on a biotin-free diet had an LC₅₀ of 5.1 g ml^–1 after 14 days compared with 76.7 g ml^–1 for larvae from a colony reared on general purpose diet. The implications for use of these proteins to confer insect resistance on transgenic plants are discussed.