Gary D. Lyon

Isolation of Potato Genes That Are Induced During an Early Stage of the Hypersensitive Response to Phytophthora infestans

Suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for sequences induced in a late-blight-resistant potato cultivar undergoing the hypersensitive response (HR). Of 100 partial cDNA sequences submitted to international DNA and protein data bases, 42 showed similarity to 35 genes, of which 31 were from plants. Of these, 13 were previously characterized as either defense-, stress-, or senescence-associated. One sequence matched (75 to 81%) all known serine palmitoyltransferases (SPTs) at the protein level. SPT catalyzes the first committed step in the synthesis of sphingolipids, important signaling molecules involved in cell differentiation and apoptosis. Putative products of other genes identified here may play a role in programmed cell death, including protein degradation, DNA degradation, metal ion chelation, and signal transduction. cDNA-amplified fragment length polymorphism was used to confirm differential expression of sequences isolated by SSH.

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans.

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea

Abstract. The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

Botrytis cinerea Perturbs Redox Processes as an Attack Strategy in Plants

Plant tissues and pathogen inoculation. Leaves of intact plants of P. vulgaris and A. thaliana were inoculated with 14-day-old conidial suspensions of B. cinerea isolate B05.10 under conditions that gave rise to spreading soft rots ; other work used detached fruits of C. annuum inoculated with another isolate . Samples for analysis were taken from within lesions, at the lesion margins and at varying distances into uninfected tissue, and compared with samples from non-inoculated controls held under the same conditions.

A risk assessment study of plant genetic transformation using Agrobacterium and implications for analysis of transgenic plants

Agrobacterium transformation systems forBrassica, Solanum andRubus, using carbenicillin, cefotaxime and ticaracillin respectively to eliminate contamination, were examined for the presence of residualAgrobacterium. The results indicated that none of the antibiotics in question, succeeded in eliminatingAgrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent thatSolanum cultures senesced and died. This may be due to the fact that four times the Minimum bactericidal concentration values (concentration to be used for elimination of contaminants in culture), for the three antibiotics, were higher than the concentrations employed in the culture medium. Contamination in shoot material decreased over 16 to 24 weeks possibly due to bacteriostatis and the use only of the apical node for further culture. The presence of the binary vector was also noted under non-selective conditions, even up to 6 months after transformation, where approx. 50% of contaminated material still harboured bacterial cells with the binary vector at levels of approx. 107 Colony forming units per gram.

Botrytis cinerea induces the formation of free radicals in fruits ofCapsicum annuum at positions remote from the site of infection

SummaryFree radical adducts of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits ofCapsicum annuum investigated 5 days after infection withBotrytis cinerea.The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.

A potato gene, erg-1, is rapidly induced by Erwinia carotovora ssp. atroseptica, Phytophthora infestans, ethylene and salicylic acid

Summary A potato gene, erg-1, was isolated from a cDNA library enriched by suppression subtractive hybridisation for sequences up-regulated one hour after inoculation of cv. Stirling leaves with Erwinia carotovora ssp. atroseptica (Eca). Erg-1 (Eca response gene 1) exhibits strong similarity to phi-1, previously reported to be up-regulated by addition of phosphate to phosphate-starved tobacco cells. Furthermore, erg-1 is similar to families of related genes in tomato, Arabidopsis, and rice, but shows no homology to sequences from animals, fungi, or prokaryotes. Northern analyses showed erg-1 to be tightly regulated and rapidly induced by Eca, and by both compatible and incompatible races of Phytophthora infestans. In addition, erg-1 is up-regulated by treatment with Erwinia carotovora culture filtrate, ethylene and salicylic acid, showing that it is regulated by more than one defence-related signalling pathway.

The biochemical basis of resistance of potato to soft rot bacteria

Evidence is presented that a number of mechanisms are involved in the resistance of commercial cultivars of potato to soft rottingErwinia spp. These include phytoalexins, phenolics, cell wall modification, and possibly proteins. The role of elicitor-active oligogalacturonides, which are released from plant cell walls by pectic enzymes, is discussed.

Responses of cells and protoplasts of Coffea arabica genotypes to partially purified culture filtrates produced by Colletotrichum kahawae

Abstract Hypocotyl-derived calli of genotypes and segregating populations of Coffea arabica, differing in susceptibility to Colletotrichum kahawae, were used to produce cell suspensions and protoplasts which were exposed to partially purified culture filtrates (PPCFs) prepared from the pathogen. The growth and viability of PPCF-treated cells and protoplasts were measured using packed cell volume, fluorescein diacetate staining and a colorimetric assay involving the tetrazolium salt MTT. Differential responses of cells and protoplasts were influenced by genotype, time of exposure and PPCF concentration. Protoplasts of resistant genotypes responded differentially from more susceptible genotypes as early as 4 h after challenge with the phytotoxin, suggesting that they were more sensitive than cell suspensions to the treatments. Protoplasts exposed to PPCFs from C. kahawae may therefore be used to screen and select genotypes resistant to, or tolerant of, coffee berry disease.

The involvement of oxygen-derived free radicals in the resistant response of potato tubers toErwinia carotovora

SummaryThe absolute requirement of oxygen for a potato tuber (cv. Pentland Crown) to display a resistant response toErwinia carotovora has been demonstrated in EPR measurements. These show that a relatively stable free radical is formed in inoculated hosts only after exposure to air. In an attempt to isolate and identify unstable free radical precursors, experiments have been conducted in which the chemical spin trap α-(4-pyridyl 1-oxide)-N-tert-butyl-nitrone (POBN) was incorporated into the tissue either at the time of inoculation or upon exposure to air. A single radical adduct was observed with spectral parameters that resemble those of a lipid-derived radical.