Overexpression and Down-Regulation of Barley Lipoxygenase<i> LOX2.2 </i>Affects Jasmonate-Regulated Genes and Aphid Fecundity

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

Genomic analyses of the Linum distyly supergene reveal convergent evolution at the molecular level

Supergenes govern multi-trait-balanced polymorphisms in a wide range of systems; however, our understanding of their origins and evolution remains incomplete. The reciprocal placement of stigmas and anthers in pin and thrum floral morphs of distylous species constitutes an iconic example of a balanced polymorphism governed by a supergene, the distyly S-locus. Recent studies have shown that the Primula and Turnera distyly supergenes are both hemizygous in thrums, but it remains unknown whether hemizygosity is pervasive among distyly S-loci. As hemizygosity has major consequences for supergene evolution and loss, clarifying whether this genetic architecture is shared among distylous species is critical. Here, we have characterized the genetic architecture and evolution of the distyly supergene in Linum by generating a chromosome-level genome assembly of Linum tenue, followed by the identification of the S-locus using population genomic data. We show that hemizygosity and thrum-specific expression of S-linked genes, including a pistil-expressed candidate gene for style length, are major features of the Linum S-locus. Structural variation is likely instrumental for recombination suppression, and although the non-recombining dominant haplotype has accumulated transposable elements, S-linked genes are not under relaxed purifying selection. Our findings reveal remarkable convergence in the genetic architecture and evolution of independently derived distyly supergenes, provide a counterexample to classic inversion-based supergenes, and shed new light on the origin and maintenance of an iconic floral polymorphism.European Research Council (ERC) 757451Swedish Research Council 2019-04452, 2018-0597

Overexpression of the aphid-induced serine protease inhibitor <i>CI2c </i>gene in barley affects the generalist green peach aphid, not the specialist bird cherry-oat aphid

<div><p>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 (<i>Hordeum vulgare</i> L.). The <i>CI2c</i> gene was earlier shown to be upregulated by herbivory of the bird cherry-oat aphid <i>(Rhopalosiphum padi</i> L.<i>)</i> 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 <i>CI2c</i> was overexpressed in barley and bioassays were carried out with <i>R</i>. <i>padi</i>. For comparison, tests were carried out with the green peach aphid (<i>Myzus persicae</i> Sulzer), for which barley is a poor host. The performance of <i>R</i>. <i>padi</i> was not different on the <i>CI2c</i>-overexpressing lines in comparison to controls in test monitoring behavior and fecundity. <i>M</i>. <i>persicae</i> preference was affected as shown in the choice test, this species moved away from control plants, but remained on the <i>CI2c</i>-overexpressing lines. <i>R</i>. <i>padi</i>-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 β-1,3-glucanase was more strongly induced by aphids in one of the <i>CI2c</i>-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 <i>R</i>.<i>padi</i>, but causes lower non-host resistance towards the generalist <i>M</i>. <i>persicae</i> in barley.</p></div

Barley defense genes against aphids

Aphids are insect pests with major importance worldwide. By feeding from plant phloem, they directly withdraw nutrients. The feeding injury is often visible in the form of leaf rolling, chlorosis, necrosis or plant deformation. Their pest status is attributed also to vectoring of plant viruses. Aphid infestation on crop plants is currently managed with the use of pesticides, which may pose a threat to the environment. An alternative approach would be to identify and characterize genetic factors contributing to aphid resistance, as well as agents inducing resistance, with the intention to use this knowledge in breeding programs. The aim of this thesis was to identify such resistance genes and characterize their involvement in plant-aphid interactions. This was accomplished using two approaches. Firstly, two putative aphid-resistance genes from barley, CI2c encoding a chymotrypsin inhibitor and LOX2.2 encoding a lipoxygenase, were transformed into Arabidopsis and/or barley and the effects of transformation were studied with regard to the performance of two aphid species. One was the specialist bird cherry-oat aphid (Rhopalosiphum padi L.), which is a pest on major cereals, and the other was a generalist, the green peach aphid (Myzus persicae Sulzer), which is a pest on plants belonging to more than 40 families. The effects of transformation were also studied concerning effects on the expression of other defense-related genes in the transgenic plants. Secondly, the effects of plant treatment with volatiles were studied with regard to induction of plant resistance, followed by tests with bird cherry-oat aphid on the treated plants. The study of overexpression of CI2c showed that the gene product transiently reduced green peach aphid fecundity on transgenic Arabidopsis, but indirectly decreased this aphid’s avoidance of barley by suppressing defense. The transformation had no effects on bird cherry-oat aphids’ behaviour or fecundity. Overexpression of LOX2.2 was shown to affect expression of other genes regulated by jasmonic acid and decreased the short-term fecundity of both the bird cherry-oat aphid and the green peach aphid on barley. The study of volatile treatments supported the idea that resistance against aphids can be induced by application of volatiles. Several defense gene sequences were induced by application of methyl salicylate, methyl jasmonate and (Z)-3-hexen-1-ol. Of the three volatiles tested, methyl jasmonate showed the greatest potential as inducing agent, causing a short-term reduction in aphid fecundity. To conclude, this thesis supports the ideas that the barley genes CI2c and LOX2.2 play a role in resistance against aphids and that moderate aphid resistance can be induced by external factors. Aphids may be directly affected by the gene product or there may be an indirect effect, caused by changes in the expression of other genes involved in plant defense. The observed negative effects on aphids were of moderate magnitude and it is proposed that acting individually, those genes are not likely to cause a strong negative effect, but they may contribute to provide resistance to aphids.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 4: Manuscript.</p

Barley defense genes against aphids

Aphids are insect pests with major importance worldwide. By feeding from plant phloem, they directly withdraw nutrients. The feeding injury is often visible in the form of leaf rolling, chlorosis, necrosis or plant deformation. Their pest status is attributed also to vectoring of plant viruses. Aphid infestation on crop plants is currently managed with the use of pesticides, which may pose a threat to the environment. An alternative approach would be to identify and characterize genetic factors contributing to aphid resistance, as well as agents inducing resistance, with the intention to use this knowledge in breeding programs. The aim of this thesis was to identify such resistance genes and characterize their involvement in plant-aphid interactions. This was accomplished using two approaches. Firstly, two putative aphid-resistance genes from barley, CI2c encoding a chymotrypsin inhibitor and LOX2.2 encoding a lipoxygenase, were transformed into Arabidopsis and/or barley and the effects of transformation were studied with regard to the performance of two aphid species. One was the specialist bird cherry-oat aphid (Rhopalosiphum padi L.), which is a pest on major cereals, and the other was a generalist, the green peach aphid (Myzus persicae Sulzer), which is a pest on plants belonging to more than 40 families. The effects of transformation were also studied concerning effects on the expression of other defense-related genes in the transgenic plants. Secondly, the effects of plant treatment with volatiles were studied with regard to induction of plant resistance, followed by tests with bird cherry-oat aphid on the treated plants. The study of overexpression of CI2c showed that the gene product transiently reduced green peach aphid fecundity on transgenic Arabidopsis, but indirectly decreased this aphid’s avoidance of barley by suppressing defense. The transformation had no effects on bird cherry-oat aphids’ behaviour or fecundity. Overexpression of LOX2.2 was shown to affect expression of other genes regulated by jasmonic acid and decreased the short-term fecundity of both the bird cherry-oat aphid and the green peach aphid on barley. The study of volatile treatments supported the idea that resistance against aphids can be induced by application of volatiles. Several defense gene sequences were induced by application of methyl salicylate, methyl jasmonate and (Z)-3-hexen-1-ol. Of the three volatiles tested, methyl jasmonate showed the greatest potential as inducing agent, causing a short-term reduction in aphid fecundity. To conclude, this thesis supports the ideas that the barley genes CI2c and LOX2.2 play a role in resistance against aphids and that moderate aphid resistance can be induced by external factors. Aphids may be directly affected by the gene product or there may be an indirect effect, caused by changes in the expression of other genes involved in plant defense. The observed negative effects on aphids were of moderate magnitude and it is proposed that acting individually, those genes are not likely to cause a strong negative effect, but they may contribute to provide resistance to aphids.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 4: Manuscript.</p

The Protease Inhibitor CI2c Gene Induced by Bird Cherry-Oat Aphid in Barley Inhibits Green Peach Aphid Fecundity in Transgenic Arabidopsis

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

Aphid numbers in choice tests.

<p>White bars represent control plants and black bars transgenic plants. Twenty adult apterous BCAs were placed in between a control and a transgenic plant in the same pot. Ten adult apterous GPA were placed within small cages on the control and on the transgenic plant in the same pot and after 24 h, the cages were opened. The total number of aphids on each genotype was counted after 5 days. Results are presented as percentage of total number of aphids counted on each plant pair. Average aphid numbers (±SE) on pair Con/CI2c 6–3 were 136.2 ± 10.2 (BCA) and 7.0 ± 1.9 (GPA) and on Con/CI2c 6–4: 120.7 ± 15.1 (BCA), respectively 22.3 ± 7.0 (GPA). Asterisks indicate significant differences (Wilcoxon matched pair test, <i>p</i>≤0.05). n = 6.</p

<i>CI2c</i> transcript abundance and inhibitory activities in transgenic lines.

<p>(A) The relative transcript abundance of the <i>CI2c</i> sequence. (B) Chymotrypsin inhibition by plant protein extracts. The results represent the average (±SE) of (A) six biological replicates, three technical replicates, (B) three biological replicates, three technical replicates. Each biological replicate consisted of primary leaf tissue from two plants, 9 days old. The transcript abundance was calculated relative to two reference genes: <i>Hsp70</i> and <i>SF427</i> and normalized to the control line set as 1.00. Chymotrypsin inhibition was normalized to the control line set as 0% inhibition. Letters indicate significant differences between lines (A: Kruskal-Wallis test, <i>p</i>≤0.05; B: one-way ANOVA followed by Tukey HSD post hoc test, <i>p</i>≤0.05).</p

Transcript abundance in barley leaves with and without BCA.

<p>Primary leaves were infested during 48 h with twenty adult apterous BCA. White bars represent uninfested plants and black bars aphid-infested plants (±SE). The transcript abundance was calculated relative to the reference genes: <i>Hsp70</i> and <i>SF427</i> and normalized to uninfested control line set as 1.00. Different letters indicate significant differences between the lines (Kruskal-Wallis test, <i>p</i>≤0.05), asterisks indicate significant difference in one genotype with or without aphids (Mann-Whitney test, *<i>p ≤</i> 0.05, **<i>p ≤</i> 0.01). Six biological replicates (with two plants each) and three technical replicates.</p