Aphids are serious pests in crop plants. In an effort to identify plant genes controlling resistance against aphids, we have here studied a protease inhibitor, CI2c in barley (Hordeum vulgare L.). The CI2c gene was earlier shown to be upregulated by herbivory of the bird cherry-oat aphid (Rhopalosiphum padi L.) in barley genotypes with moderate resistance against this aphid, but not in susceptible lines. We hypothesized that CI2c contributes to the resistance. To test this idea, cDNA encoding CI2c was overexpressed in barley and bioassays were carried out with R. padi. For comparison, tests were carried out with the green peach aphid (Myzus persicae Sulzer), for which barley is a poor host. The performance of R. padi was not different on the CI2c-overexpressing lines in comparison to controls in test monitoring behavior and fecundity. M. persicae preference was affected as shown in the choice test, this species moved away from control plants, but remained on the CI2c-overexpressing lines. R. padi-induced responses related to defense were repressed in the overexpressing lines as compared to in control plants or the moderately resistant genotypes. A putative susceptibility gene, coding for a b-1,3-glucanase was more strongly induced by aphids in one of the CI2c-overexpressing lines. The results indicate that the CI2c inhibitor in overexpressing lines affects aphid-induced responses by suppressing defense. This is of little consequence to the specialist R.padi, but causes lower non-host resistance towards the generalist M. persicae in barley.

Thiamine is a vitamin that has been shown to act as a trigger to activate plant defence and reduce pathogen and nematode infection as well as aphid settling and reproduction. We have here investigated whether thiamine treatments of seeds (i.e. seed dressing) would increase plant resistance against aphids and whether this would have different effects on a generalist than on specialist aphids. Seeds of wheat, barley, oat and pea were treated with thiamine alone or in combination with the biocontrol bacteria Pseudomonas chlororaphis MA 342 (MA 342). Plants were grown in climate chambers. The effects of seed treatment on fecundity, host acceptance and life span were studied on specialist aphids bird cherry-oat aphid (Rhopalosiphum padi L.) and pea aphid (Acyrthosiphon pisum Harris) and on the generalist green peach aphid (Myzus persicae, Sulzer). Thiamine seed treatments reduced reproduction and host acceptance of all three aphid species. The number of days to reproduction, the length of the reproductive life, the fecundity and the intrinsic rate of increase were found reduced for bird cherry-oat aphid after thiamine treatment of the cereal seeds. MA 342 did not have any effect in any of the plant-aphid combinations, except a weak decrease of pea aphid reproduction on pea. The results show that there are no differential effects of either thiamine or MA 342 seed treatments on specialist and generalist aphids and suggest that seed treatments with thiamine has a potential in aphid pest management.

Aphids are pests on many crops and depend on plant phloem sap as their food source. In an attempt to find factors improving plant resistance against aphids, we studied the effects of overexpression and down-regulation of the lipoxygenase gene LOX2.2 in barley (Hordeum vulgare L.) on the performance of two aphid species. A specialist, bird cherry-oat aphid (Rhopalosiphum padi L.) and a generalist, green peach aphid (Myzus persicae Sulzer) were studied. LOX2.2 overexpressing lines showed up-regulation of some other jasmonic acid (JA)-regulated genes, and antisense lines showed down-regulation of such genes. Overexpression or suppression of LOX2.2 did not affect aphid settling or the life span on the plants, but in short term fecundity tests, overexpressing plants supported lower aphid numbers and antisense plants higher aphid numbers. The amounts and composition of released volatile organic compounds did not differ between control and LOX2.2 overexpressing lines. Up-regulation of genes was similar for both aphid species. The results suggest that LOX2.2 plays a role in the activation of JA-mediated responses and indicates the involvement of LOX2.2 in basic defense responses.

Aphids are phloem feeders that cause large damage globally as pest insects. They induce a variety of responses in the host plant, but not much is known about which responses are promoting or inhibiting aphid performance. Here, we investigated whether one of the responses induced in barley by the cereal aphid, bird cherry-oat aphid (Rhopalosiphum padi L.) affects aphid performance in the model plant Arabidopsis thaliana L. A barley cDNA encoding the protease inhibitor CI2c was expressed in A. thaliana and aphid performance was studied using the generalist green peach aphid (Myzus persicae Sulzer). There were no consistent effects on aphid settling or preference or on parameters of life span and long-term fecundity. However, short-term tests with apterous adult aphids showed lower fecundity on three of the transgenic lines, as compared to on control plants. This effect was transient, observed on days 5 to 7, but not later. The results suggest that the protease inhibitor is taken up from the tissue during probing and weakly inhibits fecundity by an unknown mechanism. The study shows that a protease inhibitor induced in barley by an essentially monocot specialist aphid can inhibit a generalist aphid in transgenic Arabidopsis.

Steroidal glycoalkaloids (SGA) are sterol-derived neurotoxic defence substances present in several members of the Solanaceae. In the potato (Solanum tuberosum), high SGA levels may render tubers harmful for consumption. Tuber SGA levels depend on genetic factors, and can increase as a response to certain stresses and environmental conditions. To identify genes underlying the cultivar variation in tuber SGA levels, we investigated two potato cultivars differing in their SGA accumulation during wounding or light exposure; two known SGA-inducing treatments. Using microarray analysis coupled to sterol and SGA quantifications, we identified a small number of differentially expressed genes that were associated with increased SGA levels. Two of these genes, encoding distinct types of sterol Delta(24)-reductases, were by sense/antisense expression in transgenic potato plants shown to have differing roles in sterol and SGA metabolism. The results show that an increased SGA level in potato tubers during both wounding and light exposure is mediated by coordinated expression of a set of key genes in isoprenoid and steroid metabolism, and suggest that differences in this expression underlie cultivar variations in SGA levels. These results may find use within potato breeding and quality assessment.

In this study, 15 closely related barley genotypes were analyzed for the abundance of three β-1,3-glucanase transcripts immediately before and during infestation by the bird cherry-oat aphid (Rhopalosiphum padi L.). The barley lines are doubled haploid lines in backcross (BC) generations BC1 and BC2 from a cross between cultivar Lina and a wild barley accession. Previously, they have been characterized as susceptible (S) or resistant (R) to R. padi based on their ability to support nymphal growth. Here we also tested whether resistance was manifested as reduced aphid settling on the plants. Indeed, aphid numbers were lower on R than on S lines in all cases where there were significant differences between R and S lines. The choice of β-1,3-glucanase sequences is based on earlier results comparing two S and two R genotypes, suggesting that at least two of the three studied sequences are susceptibility factors. The comparisons of transcript abundance in plants with aphids showed for two of the β-1,3-glucanase sequences that there were several cases where an S genotype had significantly higher abundance than an R genotype, and in no case did an R line have significantly higher abundance than an S line. Thus, there was some support for the idea that β-1,3-glucanase sequences are susceptibility factors in the interaction between barley and R. padi.

Aphids such as the bird cherry-oat aphid (Rhopalosiphum padi L.) are serious pests in agriculture worldwide. Partially resistant barley genotypes have been produced in a breeding program for resistance to R. padi. The resistance is manifested as smaller aphids after a certain period of nymphal growth on the test plants. A large number of doubled haploid breeding lines thereby characterized as partially resistant or susceptible to R. padi are available from this breeding program. In the present study a selection of these lines, spanning four generations and 23 genotypes, was used for the evaluation of the constitutive transcript abundance of gene sequences in young barley plants. The genes were selected from an earlier microarray study where they had been identified as being up-regulated by aphids in all four barley genotypes studied or differentially up-regulated or constitutively expressed in the two resistant as compared to the two susceptible barley genotypes. The prediction for constitutive transcript abundance across the selection of genotypes was here confirmed for five out of eight investigated genes. Two of these were, as predicted, expressed at equal levels in all genotypes. Two gene sequences exhibited higher transcript abundance in resistant than in susceptible offspring; one coding for a thionin and another for a proteinase inhibitor. A lipoxygenase gene had higher transcript abundance in the susceptible lines. The study thus gives further support for putative constitutive roles of three genes previously identified as significant in the barley-R. padi interaction.

Steroidal glycoalkaloids (SGA) are toxic secondary metabolites naturally occurring in the potato, as well as in certain other Solanaceous plant species, such as tomato, eggplant and pepper. To investigate the steroidal origin of SGA biosynthesis, cut potato shoots were fed cholesterol labelled with deuterium (D) in the sterol ring structure (D-5- or D-6-labelled), or side chain (D-7-labelled), and analysed after three or five weeks. The labelled cholesterol and presence of D-labelled SGA were analysed by GC-MS and LC-MS/MS, respectively. When feeding D-labelled cholesterol solubilised in Tween-80, labelled cholesterol in free form became present in both leaves and stems, although the major part was recovered as steryl esters. Minor amounts of D-labelled SGA (alpha-solanine and alpha-chaconine) were identified in cholesterol-treated shoots, but not in blank controls, or in shoots fed D-6-27-hydroxycholesterol. Solubilising the labelled cholesterol in methyl-beta-cyclodextrin instead of Tween-80 increased the levels of labelled SGA up to 100-fold, and about 1 mole% of the labelled cholesterol was recovered as labelled SGA in potato leaves. Both side chain and ring structure D labels were retained in SGA, showing that the entire cholesterol molecule is converted to SGA. However, feeding side chain D-7-labelled cholesterol resulted in D-5-labelled SGA, indicating that two hydrogen atoms were released during formation of the SGA nitrogen-containing ring system. Feeding with D-7-sitosterol did not produce any labelled SGA, indicating that cholesterol is a specific SGA precursor. In conclusion, we have demonstrated a superior performance of methyl-beta-cyclodextrin for delivery of cholesterol in plant tissue feeding experiments, and given firm evidence for cholesterol as a specific sterol precursor of SGA in potato.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (alpha-chaconine and a-solanine) and calystegines (A(3), B-2, and B-4) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B-4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Treatment of plants with thiamine (Vitamin B1) has before been shown to activate plant defence against microorganisms. Here, we have studied the effects of thiamine treatments of plants on aphid reproduction and behaviour. The work was mainly carried out with bird cherry-oat aphid (Rhopalosiphum padi L.) on barley (Hordeum vulgare L.). Aphid population growth and aphid acceptance on plants grown from seeds soaked in a 150 mu M thiamine solution were reduced to ca. 60% of that on control plants. R. padi life span and the total number of offspring were reduced on barley plants treated with thiamine. Healthy aphids and aphids infected with the R. padi virus were similarly affected. Spraying or addition of thiamine at 150 mu M to nutrient solutions likewise resulted in reduced aphid population growth to ca. 60%, as did plant exposure to thiamine odour at 4 mM. Thiamine treatments resulted in reduced aphid population growth also when tested with grain aphid (Sitobion avenae F.) on barley and pea aphid (Acyrthosiphon pisum H.) on pea (Pisum sativum L). There was no direct effect of thiamine on aphid reproduction or thiamine odour on aphid behaviour, as evaluated using artificial diets and by olfactometer tests, respectively. Two gene sequences regulated by salicylic acid showed higher transcript abundance and one gene sequence regulated by methyl jasmonate showed lower transcript abundance in thiamine-treated plants but not in control plants after aphid infestation. These results suggest that the aphid antibiosis and antixenosis effects may be related to priming of defence, but more studies are needed to explain the effects against aphids.