Atypical molecular features of RNA silencing against the phloem-restricted polerovirus TuYV.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Plant growth-promoting bacteria Kosakonia radicincitans mediate anti-herbivore defense in Arabidopsis thaliana.

MAIN CONCLUSION: This study demonstrates that the application of the PGPB strain, Kosakonia radicincitans enhances a plant's resistance against phloem-feeding and chewing insects in Arabidopsis thaliana. The plant growth-promoting bacterial strain K. radicincitans DSM 16656 applied to A. thaliana reduced the number of phloem-feeding insects of both the specialist Brevicoryne brassicae and the generalist Myzus persicae. While weight gain of the generalist chewing insect Spodoptera exigua was reduced by 30% on A. thaliana plants treated with K. radicincitans, growth of the specialist caterpillar Pieris brassicae was not affected when compared with caterpillars from control plants. Since generalist and specialist chewing insects responded differentially to PGPB application, the implication of signaling pathways in PGPB mediated changes in plant defense was studied using two signaling pathway mutants impaired in their salicylic acid (npr1-1 mutant) or jasmonic acid (coi1-1 mutant) pathway. We found that the jasmonic acid pathway is relevant for upregulation of aliphatic glucosinolates and suppression of the chewing generalist S. exigua larval growth. Chewing from generalist P. brassicae increased glucosinolate content in A. thaliana leaves mediated via both signaling pathways. However, only in the npr1-1 mutant, which contains the highest aliphatic glucosinolate content, the P. brassicae induced further enrichment of glucosinolates, resulting in a reduction of larval growth. Effects of K. radicincitans on plant resistance could not be explained by changes in glucosinolate contents or composition. Our results demonstrate the distinct role played by K. radicincitans in suppressing insect performance in A. thaliana.

Hitchhikers, highway tolls and roadworks: the interactions of plant viruses with the phloem.

The phloem is of central importance to plant viruses, providing the route by which they spread throughout their host. Compared with virus movement in non-vascular tissue, phloem entry, exit, and long-distance translocation usually involve additional viral factors and complex virus-host interactions, probably, because the phloem has evolved additional protection against these molecular 'hitchhikers'. Recent progress in understanding phloem trafficking of endogenous mRNAs along with observations of membranous viral replication 'factories' in sieve elements challenge existing conceptions of virus long-distance transport. At the same time, the central role of the phloem in plant defences against viruses and the sophisticated viral manipulation of this host tissue are beginning to emerge.

The enemy within: phloem-limited pathogens.

The growing impact of phloem-limited pathogens on high-value crops has led to a renewed interest in understanding how they cause disease. Although these pathogens cause substantial crop losses, many are poorly characterized. In this review, we present examples of phloem-limited pathogens that include intracellular bacteria with and without cell walls, and viruses. Phloem-limited pathogens have small genomes and lack many genes required for core metabolic processes, which is, in part, an adaptation to the unique phloem environment. For each pathogen class, we present multiple case studies to highlight aspects of disease caused by phloem-limited pathogens. The pathogens presented include Candidatus Liberibacter asiaticus (citrus greening), Arsenophonus bacteria, Serratia marcescens (cucurbit yellow vine disease), Candidatus Phytoplasma asteris (Aster Yellows Witches' Broom), Spiroplasma kunkelii, Potato leafroll virus and Citrus tristeza virus. We focus on commonalities in the virulence strategies of these pathogens, and aim to stimulate new discussions in the hope that widely applicable disease management strategies can be found.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.

The green peach aphid Myzus persicae perform better on pre-infested Chinese cabbage Brassica pekinensis by enhancing host plant nutritional quality.

The green peach aphid, Myzus persicae Sulzer, is a notorious pest on vegetables, which often aggregates in high densities on crop leaves. In this study, we investigated whether M. persicae could suppress the resistance level of Chinese cabbage Brassica pekinensis. M. persicae performed better in terms of weight gain (~33% increase) and population growth (~110% increase) when feeding on previously infested (pre-infested) Chinese cabbage compared with those on non-infested plants. However, when given a choice, 64% of the aphids preferred to settle on non-infested leaves, while 29% of aphids chose pre-infested leaves that had a 2.9 times higher concentration of glucosinolates. Aphid feeding significantly enhanced the amino acid:sugar ratio of phloem sap and the absolute amino acid concentration in plant leaves. Aphid infestation significantly increased the expression levels of salicylic acid (SA) marker genes, while it had marginal effects on the expression of jasmonate marker genes. Exogenously applied SA or methyl jasmonate had no significant effects on M. persicae performance, although these chemicals increased glucosinolates concentration in plant leaves. M. persicae infestation increase amino acid:sugar ratio and activate plant defenses, but aphid performed better on pre-infested plants, suggesting that both nutrition and toxics should be considered in insect-plant interaction.

Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays.

As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.

Characterization of the Dual Subcellular Localization of Lilium LsGRP1, a Plant Class II Glycine-Rich Protein.

The defense-related gene LsGRP1 exhibits an increased level of expression in Lilium spp. after being infected by Botrytis elliptica, the fungal pathogen of lily leaf blight. In this study, the expression profile of the LsGRP1 protein (a plant class II glycine-rich protein) was characterized biochemically and its subcellular localization in lily leaves was evaluated using immunohistochemistry, enhanced green fluorescent protein (EGFP) imaging, and protein extraction analysis. Using an LsGRP1-specific antibody, LsGRP1 was found to be most abundant in epidermal cells and phloem tissues. Leaves from lily plants at different growth stages demonstrated similar levels of 14- and 16-kDa LsGRP1 and a decreased amount of 23-kDa LsGRP1 at the senescence stage. LsGRP1-EGFP imaging and protein extraction assays revealed that 14-kDa LsGRP1 was located in the plasma membrane whereas 16- and 23-kDa LsGRP1 was weakly bound to the cell wall. The time course analyses of LsGRP1 expression in response to salicylic acid treatment or B. elliptica infection showed an increased accumulation of 14- and 23-kDa LsGRP1 over time. Because 23-kDa LsGRP1 could be detected by an ubiquitin antibody, conversion of 14-kDa to 23-kDa LsGRP1 via mono-ubiquitination was presumed, which is a phenomenon that has not been reported for a plant class II glycine-rich protein.

Transgenic expression of a functional fragment of harpin protein Hpa1 in wheat induces the phloem-based defence against English grain aphid.

The harpin protein Hpa1 has multiple beneficial effects in plants, promoting plant growth and development, increasing crop yield, and inducing resistance to pathogens and insect pests. For these effects, the 10-40 residue fragment (Hpa110₋42) isolated from the Hpa1 sequence is 1.3- to 7.5-fold more effective than the full-length protein. Here it is reported that the expression of Hpa110₋42 under the direction of an insect-induced promoter induces the phloem-based defence to English grain aphid, a dominant species of wheat aphids. The expression of Hpa110₋42 was found to compromise the colonization preference of aphids on the plant and further inhibit aphid reproduction in leaf colonies. In Hpa110₋42-expressing wheat lines, moreover, aphid feeding from the phloem was repressed in correlation with the phloem-based defence. This defensive mechanism was shown as enhanced expression of wheat genes encoding phloem lectin proteins (PP2-A1 and PP2-A2) and ß-1,3-glucan synthase-like enzymes (GSL2, GSL10, and GSL12). Both PP2-A and ß-1,3-glucan formed high molecular mass polymers to block phloem sieve plate pores and therefore impede aphid feeding from the phloem. However, the phloem-based defence was impaired by treating plants with ethylene signalling inhibitors, suggesting the requirement for the ethylene signalling pathway. In addition, if Hpa110₋42-expressing plants were subjected to attack by a small number of aphids, they newly acquired agriculturally beneficial characters, such as enhanced vegetative growth and increased tiller numbers and grain output values. These results suggest that the defensive and developmental roles of Hpa110₋42 can be integrated into the germplasm of this agriculturally significant crop.

Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

Long distance root-shoot signalling in plant-insect community interactions.

Plants mediate interactions between insects, including leaf- and root-feeders; yet the underlying mechanisms and connection with ecological theory remain unresolved. In this review, based on novel insights into long-distance (i.e., leaf-leaf, root-shoot) defence signalling, we explore the role of phytohormones in driving broad-scale patterns of aboveground-belowground interactions that can be extrapolated to general plant-insect relationships. We propose that the outcome of intra-feeding guild interactions is generally negative due to induction of similar phytohormonal pathways, whereas between-guild interactions are often positive due to negative signal crosstalk. However, not all outcomes could be explained by feeding guild; we argue that future studies should target ecologically representative plant-insect systems, distinguish subguilds, and include plant growth hormones to improve our understanding of plant-mediated interactions.

Multiple phytohormone signals control the transcriptional response to soybean aphid infestation in susceptible and resistant soybean plants.

The soybean aphid (Aphis glycines) is a major phloem-feeding pest of soybean (Glycine max). A. glycines feeding can cause the diversion of photosynthates and transmission of plant viruses, resulting in significant yield losses. In this study, we used oligonucleotide microarrays to characterize the long-term transcriptional response to soybean aphid colonization of two related soybean cultivars, one with the Rag1 aphid-resistance gene and one aphid-susceptible cultivar (without Rag1). Transcriptome profiles were determined after 1 and 7 days of aphid infestation. Our results revealed a susceptible response involving hundreds of transcripts, whereas only one transcript changed in the resistant response to aphids. This nonexistent resistance response might be explained by the fact that many defense-related transcripts are constitutively expressed in resistant plants, whereas these same genes are activated in susceptible plants only during aphid infestation. Analysis of phytohormone-related transcripts in the susceptible response showed different hormone profiles for the two time points, and suggest that aphids are able to suppress hormone signals in susceptible plants. A significant activation of abscissic acid, normally associated with abiotic stress responses, at day 7, might be a decoy strategy implemented by the aphid to suppress effective salicylic acid- and jasmonate-related defenses.

Localization of phenolics in phloem parenchyma cells of Norway spruce (Picea abies).

Norway spruce (Picea abies) bark contains specialized phloem parenchyma cells that swell and change their contents upon attack by the bark beetle Ips typographus and its microbial associate, the blue stain fungus Ceratocystis polonica. These cells exhibit bright autofluorescence after treatment with standard aldehyde fixatives, and so have been postulated to contain phenolic compounds. Laser microdissection of spruce bark sections combined with cryogenic NMR spectroscopy demonstrated significantly higher concentrations of the stilbene glucoside astringin in phloem parenchyma cells than in adjacent sieve cells. After infection by C. polonica, the flavonoid (+)-catechin also appeared in phloem parenchyma cells and there was a decrease in astringin content compared to cells from uninfected trees. Analysis of whole-bark extracts confirmed the results obtained from the cell extracts and revealed a significant increase in dimeric stilbene glucosides, both astringin and isorhapontin derivatives (piceasides A to H), in fungus-infected versus uninfected bark that might explain the reduction in stilbene monomers. Phloem parenchyma cells thus appear to be a principal site of phenolic accumulation in spruce bark.

Histological examination of horse chestnut infection by Pseudomonas syringae pv. aesculi and non-destructive heat treatment to stop disease progression.

Since its emergence in Northwest Europe as a pathogen that infects trunks and branches of Aesculus spp. (the horse chestnuts) approximately one decade ago, Pseudomonas syringae pv. aesculi has rapidly established itself as major threat to these trees. Infected trees exhibit extensive necrosis of phloem and cambium, which can ultimately lead to dieback. The events after host entry leading to extensive necrosis are not well documented. In this work, the histopathology of this interaction is investigated and heat-treatment is explored as method to eradicate bacteria associated with established infections. The early wound-repair responses of A. hippocastanum, both in absence and presence of P. s. pv. aesculi, included cell wall lignification by a distinct layer of phloem and cortex parenchyma cells. The same cells also deposited suberin lamellae later on, suggesting this layer functions in compartmentalizing healthy from disrupted tissues. However, monitoring bacterial ingress, its construction appeared inadequate to constrain pathogen spread. Microscopic evaluation of bacterial dispersal in situ using immunolabelling and GFP-tagging of P. s. pv. aesculi, revealed two discriminative types of bacterial colonization. The forefront of lesions was found to contain densely packed bacteria, while necrotic areas housed bacterial aggregates with scattered individuals embedded in an extracellular matrix of bacterial origin containing alginate. The endophytic localization and ability of P. s. pv aesculi to create a protective matrix render it poorly accessible for control agents. To circumvent this, a method based on selective bacterial lethality at 39 °C was conceived and successfully tested on A. hippocastanum saplings, providing proof of concept for controlling this disease by heat-treatment. This may be applicable for curing other tree cankers, caused by related phytopathogens.

Identification and characterization of resistance to cowpea aphid (Aphis craccivora Koch) in Medicago truncatula.

BACKGROUND: Cowpea aphid (CPA; Aphis craccivora) is the most important insect pest of cowpea and also causes significant yield losses in other legume crops including alfalfa, beans, chickpea, lentils, lupins and peanuts. In many of these crops there is no natural genetic resistance to this sap-sucking insect or resistance genes have been overcome by newly emerged CPA biotypes. RESULTS: In this study, we screened a subset of the Medicago truncatula core collection of the South Australian Research and Development Institute (SARDI) and identified strong resistance to CPA in a M. truncatula accession SA30199, compared to all other M. truncatula accessions tested. The biology of resistance to CPA in SA30199 plants was characterised compared to the highly susceptible accession Borung and showed that resistance occurred at the level of the phloem, required an intact plant and involved a combination of antixenosis and antibiosis. Quantitative trait loci (QTL) analysis using a F2 population (n = 150) from a cross between SA30199 and Borung revealed that resistance to CPA is controlled in part by a major quantitative trait locus (QTL) on chromosome 2, explaining 39% of the antibiosis resistance. CONCLUSIONS: The identification of strong CPA resistance in M. truncatula allows for the identification of key regulators and genes important in this model legume to give effective CPA resistance that may have relevance for other legume crops. The identified locus will also facilitate marker assisted breeding of M. truncatula for increased resistance to CPA and potentially other closely related Medicago species such as alfalfa.

Response of living tissues of Pinus sylvestris to the saprotrophic biocontrol fungus Phlebiopsis gigantea.

The saprotrophic fungus Phlebiopsis gigantea has been used for several years as a biocontrol agent against the conifer pathogen Heterobasidion annosum. Although the effectiveness of P. gigantea in biocontrol has been shown empirically, the long-term effect on living conifer trees as well as the mechanism underlying its antagonistic activity is still unknown. An additional concern is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues. By using a combination of histochemical, molecular and transcript profiling (454 sequencing), we investigated under in vitro conditions the necrotrophic capability of P. gigantea and induced localized resistance as a mechanism for its biocontrol action. Pinus sylvestris seedlings (10 years old) were challenged on the xylem surface with P. gigantea or H. annosum. Both fungi provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had more lignified cells. The 454 sequencing revealed elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. The results suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P. gigantea, and it has a limited necrotrophic capability compared with H. annosum.

A single gene, AIN, in Medicago truncatula mediates a hypersensitive response to both bluegreen aphid and pea aphid, but confers resistance only to bluegreen aphid.

Biotic stress in plants frequently induces a hypersensitive response (HR). This distinctive reaction has been studied intensively in several pathosystems and has shed light on the biology of defence signalling. Compared with microbial pathogens, relatively little is known about the role of the HR in defence against insects. Reference genotype A17 of Medicago truncatula Gaertn., a model legume, responds to aphids of the genus Acyrthosiphon with necrotic lesions resembling a HR. In this study, the biochemical nature of this response, its mode of inheritance, and its relationship with defence against aphids were investigated. The necrotic lesion phenotype and resistance to the bluegreen aphid (BGA, Acyrthosiphon kondoi Shinji) and the pea aphid (PA, Acyrthosiphon pisum (Harris)) were analysed using reference genotypes A17 and A20, their F(2) progeny and recombinant inbred lines. BGA-induced necrotic lesions co-localized with the production of H(2)O(2), consistent with an oxidative burst widely associated with hypersensitivity. This HR correlated with stronger resistance to BGA in A17 than in A20; these phenotypes cosegregated as a semi-dominant gene, AIN (Acyrthosiphon-induced necrosis). In contrast to BGA, stronger resistance to PA in A17, compared with A20, did not cosegregate with a PA-induced HR. The AIN locus resides in a cluster of sequences predicted to encode the CC-NBS-LRR subfamily of resistance proteins. AIN-mediated resistance presents a novel opportunity to use a model plant and model aphid to study the role of the HR in defence responses to phloem-feeding insects.

A temporal analysis of antioxidative defense responses in the phloem of Picea abies after attack by Ips typographus.

The temporal gradation of antioxidants was investigated on the phloem tissue of Norway spruce [Picea abies (L.) Karst.] in response to weather conditions and colonization levels of Ips typographus L. (Col., Scolytidae). Two weeks after pheromone dispensers were placed on trees, the initial reaction of Norway spruce to bark beetle attack resulted in moderately lowered levels of total glutathione (tGSH) and total cysteine. Likewise, the total ascorbic acid dropped slightly below the control levels, whereas the concentration of dehydroascorbic acid increased in comparison to the first sampling date. This transient degradation and oxidation of the glutathione and ascorbate system was accompanied by moderately increased concentrations of total phenolics. One month later, the shift in antioxidant balance after moderate attack differed quantitatively from the reaction after massive attack. An intensification of antioxidant defense occurred within moderately affected bark. Total cysteine and tGSH contents were markedly raised, whereas the concentrations of total ascorbic acid and total phenolics were slightly increased by moderate attack. On the other hand, massive bark beetle colonization caused a strong decrease in tGSH and total phenolics, whereas total cysteine and total ascorbic acid values remained at control level. Dependent upon the intensity and the success of the attack, a progressive degradation of antioxidants was determined at later sampling dates, which was accompanied by an obvious oxidation of the ascorbate and glutathione pools. With an unsuccessful defense upon massive attack, the thiols and total phenolics did not reach a new steady state, but deteriorated until the end of the brood beetles' development. In contrast, the dynamic antioxidative response within the moderately affected trees indicated an acclimation stage in the middle of July. It was characterized by a higher accumulation of tGSH, total ascorbic acid and total phenolics as well as a more reduced redox state of glutathione. A sequence of changes in the endogenous levels of antioxidant defense molecules in the bark beetle-affected Norway spruce showed consistency with the general ecophysiological stress-response concept, and provided important avenues for evaluating the role and effectiveness of antioxidants in systemic acquired resistance against the complex interactive effects of bark beetle attack and environmental factors.