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Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana a
b
bc
b
Magdalene Kutyniok , Andrea Viehhauser , Marc Oliver Vogel , Karl-Josef Dietz & Caroline Müller
a
a
Department of Chemical Ecology; Bielefeld University; Bielefeld, Germany
b
Department of Plant Biochemistry and Physiology; Bielefeld University; Bielefeld, Germany
c
Proteome- and Metabolome Research; Bielefeld University; Bielefeld, Germany Accepted author version posted online: 31 Oct 2014.Published online: 24 Dec 2014.
Click for updates To cite this article: Magdalene Kutyniok, Andrea Viehhauser, Marc Oliver Vogel, Karl-Josef Dietz & Caroline Müller (2014) Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana, Plant Signaling & Behavior, 9:11, e976113, DOI: 10.4161/15592324.2014.976113 To link to this article: http://dx.doi.org/10.4161/15592324.2014.976113
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SHORT COMMUNICATION Plant Signaling & Behavior 9:11, e976113; December 1, 2014; © 2014 Taylor & Francis Group, LLC
Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana €ller1,* Magdalene Kutyniok1, Andrea Viehhauser2, Marc Oliver Vogel2,3, Karl-Josef Dietz2, and Caroline Mu 1
Department of Chemical Ecology; Bielefeld University; Bielefeld, Germany; 2Department of Plant Biochemistry and Physiology; Bielefeld University; Bielefeld, Germany; 3 Proteome- and Metabolome Research; Bielefeld University; Bielefeld, Germany
Keywords: aphids, gene expression, induction, nematodes, signaling
Downloaded by [Washington University in St Louis] at 00:20 21 January 2015
Abbreviations: dpi, days post infestation; JA, jasmonic acid; SA, salicylic acid; PR, pathogenesis related.
Plants are often simultaneously infested by several herbivores at the shoots and roots. Recent results revealed that the model plant Arabidopsis thaliana shows highly challenge-specific local and systemic responses to individual and simultaneous attacks of shoot-infesting aphids and root-infesting nematodes at the metabolome level.1 Here, we present the corresponding transcriptional changes in plants treated with Brevicoryne brassicae aphids and Heterodera schachtii nematodes individually and in combination. Overall, shoots were much less responsive than roots. Gene expression in shoots and roots was mainly altered by aphids. Nematode infestation alone had only little effect, but nematodes modified the transcript accumulation response to aphids particularly in the roots. The responding genes are involved in plant defense cascades, signaling, oxidation-reduction processes, as well as primary and secondary metabolism and degradation. These changes in transcription may become relevant for the herbivores when they are translated into changes in host plant quality.
Plants often face herbivore attacks on shoots and roots simultaneously, leading to fine-tuned plant responses and necessitating signaling crosstalk.2–4 Important agricultural pests such as shootsucking aphids and cyst and root-knot nematodes both parasitize on the vascular system and may therefore modify the host’s response in a similar way.4 Aphid- and nematode-induced changes in gene expression have been studied in the attacked organ (shoots or roots)5–10 but little is known about systemic effects and the interaction response to different herbivores. To better understand the impact of herbivores on the plant and their interplay when sharing a host, transcriptome analyses of the local and systemic plant tissue (shoots/roots) to individual and combined aphid and nematode feeding are needed. Changes in transcript accumulation often serve as sensitive indicators for activation of involved signaling pathways and identification of responsive gene ontology groups and target genes. Recently, we investigated the early metabolic responses of shoots and roots of Arabidopsis thaliana (Col-0) (Brassicaceae) to low feeding pressure, characteristic for the initial phase of infestation, of shoot-infesting cabbage aphids [Brevicoryne brassicae L. (Hemiptera: Aphididae), 10 aphids per plant, A] and/or rootinfesting sugar beet nematodes [Heterodera schachtii (Schmidt) (Tylenchida: Heteroderidae), inoculation of 1,000 nematodes in soil, N].1 Neither primary metabolites nor phytohormone levels were modified due to herbivory at 3 dpi. However, aphids
induced a shift in the shoot metabolic fingerprints and glucosinolate concentrations, characteristic defense compounds of the Brassicaceae, mainly in shoots but to some degree also in roots. In contrast, nematodes had little effect on the chemical composition both locally and systemically. To complement these data, here we analyzed the gene expression of plants treated in an identical experimental design as in Kutyniok and M€ uller.1 Three dpi, shoots and roots, respectively, were pooled from 3 plants of each of the 4 treatments (A, N, AN, and uninfested control plants, C), collecting 3 independent replicates per treatment. Samples were frozen in liquid nitrogen and homogenized. RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality was surveyed by Nanodrop analysis (ND-1000, PEQLAB Biotechnologie GmbH, Erlangen, Germany). Whole genome ATH1 arrays (Affymetrix; Santa Clara, CA, USA) were used to study the transcript levels of approximately 24,000 genes. Hybridization of the chips was done by Kompetenzzentrum Fluoreszente Bioanalytik (Regensburg, Germany). Acquired raw data were normalized according to the RMA algorithm11 using the ROBIN package12 in R.13 Transcript levels of C, A, N, and AN tissues were compared with t-tests. Gene annotation was done using MapMan.14 The individual and combined herbivore treatments altered the transcript levels to a different extent, with roots being more responsive than shoots. In the shoots, significant changes in
*Correspondence to: Caroline M€ uller; Email: [email protected] Submitted: 07/26/2014; Accepted: 08/08/2014 http://dx.doi.org/10.4161/15592324.2014.976113
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Plant Signaling & Behavior
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Figure 1. Significant changes of transcript abundance in (A) shoots and (B) roots in response to treatments with aphids (A, blue), nematodes (N, yellow) or both herbivores (AN) in combination (red) in comparison to controls at 3 dpi and between the 3 treatments in (C) shoots and (D) roots of Arabidopsis thaliana analyzed with whole genome microarrays. Total number of genes with changed transcript levels and number of up- (") and downregulated (#) genes are indicated. The cut-off for differentially regulated genes was set to the adjusted P-value 0.05 (according to Benjamini and Hochberg25) and a log fold change of ¡0.5 (downregulation) or 0.5 (upregulation) (N D 3).
transcription occurred only in 40 genes (Fig. 1A and C). A and AN treatments mostly up-regulated the expression of a few genes with only some overlap between the effects caused by each treatment when compared to control plants (Fig. 1A). In contrast to our results, most other studies found more genes to be affected and mainly down-regulated by aphid feeding6,7,10,15, but see16. This can most likely be attributed to varying experimental conditions such as differences in induction duration, higher numbers of aphids used for infestation and analyses of other A. thaliana ecotypes or other aphid species.6,7,15 Nematode infestation caused no transcriptional changes in the systemic shoots in accordance with the lack in effects on metabolites and phytohormones reported earlier.1 When comparing the changes in shoot gene expression between the 3 herbivore treatments, no difference was
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found between A and AN plants (Fig. 1C), but 37 genes were differentially regulated by A and AN compared to N plants with an overlap of 14 genes. Thus, although exclusive nematode infestation did not affect shoot gene expression, nematodes influenced the plant response to aboveground herbivory to some extent. Genes affected in the shoots were related to different levels of plant defense cascades, such as PR-protein coding genes, R-genes involved in gene-to-gene resistance, and genes related to oxidation-reduction processes (Table S1). Furthermore, genes involved in JA metabolism and signaling were induced, while SA-related genes seemed not to be affected in contrast to findings by Kusnierczyk et al.15 Moreover, genes relevant for primary and secondary metabolite biosynthesis, particularly of glucosinolates, and genes connected to myrosinase enzymes, were induced by A
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and AN treatments. Expression of 2 of these genes (At2g24850, At1g52000) had been also found to be elevated in response to B. brassicae earlier7,10,15 while transcript levels of some of these genes were reduced by attack of a generalist aphid6 (Table S1). Interestingly, A and AN treatments also affected processes such as macromolecule organization, organonitrogen compound processes and proteolysis. Protein and amino acid degradation and nitrogen re-assimilation may be plant strategies to generate energy during aphid attack.10 Aphids can induce changes in the allocation of primary metabolites, particularly amino acids, for their own purpose.17,18 In the roots, only one gene (At5g05340), coding for a peroxidase, of the 90 genes affected by the herbivore treatments compared to the control was upregulated by the N treatment, which also responded to AN (Fig. 1B). This gene shows also increased expression in H. schachtii syncytia8 and in A. thaliana leaves following infestation with a generalist aphid6 (Table S2). The seemingly low responsiveness of root genes to nematode infestation may be due to a dilution effect when analyzing entire roots in contrast to the exclusive analysis of syncytia.8 In roots of younger plants, 128 genes with altered transcript levels caused by a more pronounced nematode infestation have been reported.5 Aphid feeding alone and in combination with nematodes evoked pronounced changes in gene expression in the roots compared to control plants, with distinct responses to A and AN infestation (Fig. 1B) as well as N versus AN (Fig. 1D). Our results support an earlier finding that B. brassicae alters root infestation rates by H. schachtii but not vice versa1,19, but see20,21. As in shoots, genes involved in diverse important stress responses such as oxidation-reduction processes, signaling events and several genes coding for transcription factors were up-regulated in the roots (Table S1). Furthermore, genes linked to cell References 1. Kutyniok M, M€ uller C. Crosstalk between above–and belowground herbivores is mediated by minute metabolic responses of the host Arabidopsis thaliana. J Exp Bot 2012; 63:6199-210; PMID: 23045608; http://dx. doi.org/10.1093/jxbers/274 2. Maffei ME, Mithofer A, Boland W. Before gene expression: early events in plant-insect interaction. Trends Plant Sci 2007; 12:310-6; PMID: 17596996; http://dx.doi.org/10.1016/j.tplants.2007.06.001 3. Sutter R, M€ uller C. Mining for treatment-specific and general changes in target compounds and metabolic fingerprints in response to herbivory and phytohormones in Plantago lanceolata. New Phytol 2011; 191:106982; PMID: 21592133; http://dx.doi.org/10.1111/ j.1469-8137.2011.03768.x 4. Soler R, Erb M, Kaplan I. Long distance root–shoot signalling in plant–insect community interactions. Trends Plant Sci 2013; 18:149-56; PMID: 22989699; http://dx.doi.org/10.1016/j.tplants.2012.08.010 5. Puthoff DP, Nettleton D, Rodermel SR, Baum TJ. Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant J 2003; 33:911-21; PMID: 12609032 6. de Vos M, van Oosten VR, van Poecke RM, van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ, Metraux JP, Van Loon LC, et al. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol Plant Microbe Interact 2005; 18:923-37; PMID: 16167763; http://dx.doi.org/10.1094/MPMI18-0923
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wall processes and secondary metabolite biosynthesis (glucosinolates, flavonoids, and one terpene biosynthesis gene) and degradation, potentially altering plant susceptibility to phytoparasitic nematodes,22,23 were influenced by A and AN treatments. Moreover, genes with responsiveness to water deprivation were affected, most likely because aphids withdraw high amounts of solutes and can induce osmotic stress in plants.10 Several of the genes regulated by AN treatment with importance for root or cell development, organization and growth were also differentially expressed in H. schachtii syncytia8 but some responded in an opposite direction. In summary, the effects on gene expression found here indicate that aphids alone and in combination with nematodes induce highly challenge-specific plant responses realized at diverse mechanistic defense levels even at low numbers of infestation. Aphids may affect systemic tissue due to strong sink capacities.24 Exclusive nematode infestation at an early time point of interaction with the host does not lead to relevant changes of gene expression but nematode presence alters the plant gene expression response to aphids especially in the roots. Future studies should address the question whether gene expression responses are enhanced when plants are attacked by higher infestation levels of aphids in combination with nematodes. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Supplemental Material
Supplemental data for this article can be accessed on the publisher’s website.
7. Kusnierczyk A, Winge P, Midelfart H, Armbruster WS, Rossiter JT, Bones AM. Transcriptional responses of Arabidopsis thaliana ecotypes with different glucosinolate profiles after attack by polyphagous Myzus persicae and oligophagous Brevicoryne brassicae. J Exp Bot 2007; 58:2537-52; PMID: 17545220 8. Szakasits D, Heinen P, Wieczorek K, Hofmann J, Wagner F, Kreil DP, Sykacek P, Grundler FM, Bohlmann H. The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant J 2009; 57:771-84; PMID: 18980640; http://dx.doi.org/10.1111/j.1365-313X.2008.03727.x 9. Kusnierczyk A, Tran DHT, Winge P, Jørstad TS, Reese JC, Troczy nska J, Bones AM. Testing the importance of jasmonate signalling in induction of plant defences upon cabbage aphid (Brevicoryne brassicae) attack. BMC Genomics 2011; 12:423; PMID: 21854623; http://dx.doi.org/10.1186/1471-2164-12423 10. Barah P, Winge P, Kusnierczyk A, Tran DH, Bones AM. Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection. PloS One 2013; 8:e58987; PMID: 23536844; http://dx.doi. org/10.1371/journal.pone.0058987 11. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of affymetrix GeneChip probe level data. Nucleic Acids Res 2003; 31:e15; PMID: 12582260 12. Lohse M, Nunes-Nesi A, Kr€uger P, Nagel A, Hannemann J, Giorgi FM, Childs L, Osorio S, Walther D, Selbig J, et al. Robin: an intuitive wizard application for R-based expression microarray quality assessment
Plant Signaling & Behavior
13.
14.
15.
16.
17.
18.
and analysis. Plant Physiol 2010; 153:642-51; PMID: 20388663; http://dx.doi.org/10.1104/pp.109.152553 R Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 2010 Thimm O, Bl€asing O, Gibon Y, Nagel A, Meyer S, Kr€uger P, Selbig J, M€ uller LA, Rhee SY, Stitt M. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 2004; 37:914-39; PMID: 14996223 Kusnierczyk A, Winge P, Jørstad TS, Troczy nska J, Rossiter JT, Bones AM. Towards global understanding of plant defence against aphids–timing and dynamics of early Arabidopsis defence responses to cabbage aphid (Brevicoryne brassicae) attack. Plant Cell Environ 2008; 31:1097-115; PMID: 18433442; http://dx.doi. org/10.1111/j.1365-3040.2008.01823.x Couldridge C, Newbury H, Ford-Lloyd B, Bale J, Pritchard J. Exploring plant responses to aphid feeding using a full Arabidopsis microarray reveals a small number of genes with significantly altered expression. Bull Entomol Res 2007; 97:523-32; PMID: 17916270 Sandstr€om J, Telang A, Moran N. Nutritional enhancement of host plants by aphids–a comparison of three aphid species on grasses. J Insect Physiol 2000; 46:33-40; PMID: 12770256; http://dx.doi.org/ 10.1016/S0022-1910(99)00098-0 Girousse C, Moulia B, Silk W, Bonnemain J-L. Aphid infestation causes different changes in carbon and nitrogen allocation in alfalfa stems as well as different
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21. Hol WHG, de Boer W, Termorshuizen AJ, Meyer KM, Schneider JH, van Der Putten WH, van Dam NM. Heterodera schachtii nematodes interfere with aphid-plant relations on Brassica oleracea. J Chem Ecol 2013; 39:1193-203; PMID: 24014097; http://dx.doi. org/10.1007/s10886-013-0338-4 22. Potter MJ, Vanstone VA, Davies KA, Kirkegaard JA, Rathjen AJ. Reduced susceptibility of Brassica napus to Pratylenchus neglectus in plants with elevated root levels of 2-phenylethyl glucosinolate. J Nematol 1999; 31:291-8; PMID: 19270899 23. Jones JT, Furlanetto C, Phillips MS. The role of flavonoids produced in response to cyst nematode infection
of Arabidopsis thaliana. Nematology 2007; 9:671-7; http://dx.doi.org/10.1163/156854107782024875 24. Kaplan I, Sardanelli S, Rehill BJ, Denno RF. Toward a mechanistic understanding of competition in vascularfeeding herbivores: an empirical test of the sink competition hypothesis. Oecologia 2011; 166:627-36; PMID: 21181415; http://dx.doi.org/10.1007/s00442010-1885-9 25. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B Met 1995; 57:289-300; http://www.jstor.orgstable2346101">http:www.jstor. orgstable2346101
Downloaded by [Washington University in St Louis] at 00:20 21 January 2015
inhibitions of longitudinal and radial expansion. Plant Physiol 2005; 137:1474-84; PMID: 15778456 19. Kutyniok M, M€ uller C. Plant-mediated interactions between shoot-feeding aphids and root-feeding nematodes depend on nitrate fertilization. Oecologia 2013; 173:1367-77; PMID: 23836091; http://dx.doi.org/ 10.1007/s00442-013-2712-x 20. Hol WHG, de Boer W, Termorshuizen AJ, Meyer KM, Schneider JHM, van Dam NM, van Veen JA, van der Putten WH. Reduction of rare soil microbes modifies plant-herbivore interactions. Ecol Lett 2010; 13:292-301; PMID: 20070364; http://dx.doi.org/ 10.1111/j.1461-0248.2009.01424.x
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Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana a
b
bc
b
Magdalene Kutyniok , Andrea Viehhauser , Marc Oliver Vogel , Karl-Josef Dietz & Caroline Müller
a
a
Department of Chemical Ecology; Bielefeld University; Bielefeld, Germany
b
Department of Plant Biochemistry and Physiology; Bielefeld University; Bielefeld, Germany
c
Proteome- and Metabolome Research; Bielefeld University; Bielefeld, Germany Accepted author version posted online: 31 Oct 2014.Published online: 24 Dec 2014.
Click for updates To cite this article: Magdalene Kutyniok, Andrea Viehhauser, Marc Oliver Vogel, Karl-Josef Dietz & Caroline Müller (2014) Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana, Plant Signaling & Behavior, 9:11, e976113, DOI: 10.4161/15592324.2014.976113 To link to this article: http://dx.doi.org/10.4161/15592324.2014.976113
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SHORT COMMUNICATION Plant Signaling & Behavior 9:11, e976113; December 1, 2014; © 2014 Taylor & Francis Group, LLC
Local and systemic transcriptional responses to crosstalk between above- and belowground herbivores in Arabidopsis thaliana €ller1,* Magdalene Kutyniok1, Andrea Viehhauser2, Marc Oliver Vogel2,3, Karl-Josef Dietz2, and Caroline Mu 1
Department of Chemical Ecology; Bielefeld University; Bielefeld, Germany; 2Department of Plant Biochemistry and Physiology; Bielefeld University; Bielefeld, Germany; 3 Proteome- and Metabolome Research; Bielefeld University; Bielefeld, Germany
Keywords: aphids, gene expression, induction, nematodes, signaling
Downloaded by [Washington University in St Louis] at 00:20 21 January 2015
Abbreviations: dpi, days post infestation; JA, jasmonic acid; SA, salicylic acid; PR, pathogenesis related.
Plants are often simultaneously infested by several herbivores at the shoots and roots. Recent results revealed that the model plant Arabidopsis thaliana shows highly challenge-specific local and systemic responses to individual and simultaneous attacks of shoot-infesting aphids and root-infesting nematodes at the metabolome level.1 Here, we present the corresponding transcriptional changes in plants treated with Brevicoryne brassicae aphids and Heterodera schachtii nematodes individually and in combination. Overall, shoots were much less responsive than roots. Gene expression in shoots and roots was mainly altered by aphids. Nematode infestation alone had only little effect, but nematodes modified the transcript accumulation response to aphids particularly in the roots. The responding genes are involved in plant defense cascades, signaling, oxidation-reduction processes, as well as primary and secondary metabolism and degradation. These changes in transcription may become relevant for the herbivores when they are translated into changes in host plant quality.
Plants often face herbivore attacks on shoots and roots simultaneously, leading to fine-tuned plant responses and necessitating signaling crosstalk.2–4 Important agricultural pests such as shootsucking aphids and cyst and root-knot nematodes both parasitize on the vascular system and may therefore modify the host’s response in a similar way.4 Aphid- and nematode-induced changes in gene expression have been studied in the attacked organ (shoots or roots)5–10 but little is known about systemic effects and the interaction response to different herbivores. To better understand the impact of herbivores on the plant and their interplay when sharing a host, transcriptome analyses of the local and systemic plant tissue (shoots/roots) to individual and combined aphid and nematode feeding are needed. Changes in transcript accumulation often serve as sensitive indicators for activation of involved signaling pathways and identification of responsive gene ontology groups and target genes. Recently, we investigated the early metabolic responses of shoots and roots of Arabidopsis thaliana (Col-0) (Brassicaceae) to low feeding pressure, characteristic for the initial phase of infestation, of shoot-infesting cabbage aphids [Brevicoryne brassicae L. (Hemiptera: Aphididae), 10 aphids per plant, A] and/or rootinfesting sugar beet nematodes [Heterodera schachtii (Schmidt) (Tylenchida: Heteroderidae), inoculation of 1,000 nematodes in soil, N].1 Neither primary metabolites nor phytohormone levels were modified due to herbivory at 3 dpi. However, aphids
induced a shift in the shoot metabolic fingerprints and glucosinolate concentrations, characteristic defense compounds of the Brassicaceae, mainly in shoots but to some degree also in roots. In contrast, nematodes had little effect on the chemical composition both locally and systemically. To complement these data, here we analyzed the gene expression of plants treated in an identical experimental design as in Kutyniok and M€ uller.1 Three dpi, shoots and roots, respectively, were pooled from 3 plants of each of the 4 treatments (A, N, AN, and uninfested control plants, C), collecting 3 independent replicates per treatment. Samples were frozen in liquid nitrogen and homogenized. RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality was surveyed by Nanodrop analysis (ND-1000, PEQLAB Biotechnologie GmbH, Erlangen, Germany). Whole genome ATH1 arrays (Affymetrix; Santa Clara, CA, USA) were used to study the transcript levels of approximately 24,000 genes. Hybridization of the chips was done by Kompetenzzentrum Fluoreszente Bioanalytik (Regensburg, Germany). Acquired raw data were normalized according to the RMA algorithm11 using the ROBIN package12 in R.13 Transcript levels of C, A, N, and AN tissues were compared with t-tests. Gene annotation was done using MapMan.14 The individual and combined herbivore treatments altered the transcript levels to a different extent, with roots being more responsive than shoots. In the shoots, significant changes in
*Correspondence to: Caroline M€ uller; Email: [email protected] Submitted: 07/26/2014; Accepted: 08/08/2014 http://dx.doi.org/10.4161/15592324.2014.976113
www.landesbioscience.com
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Figure 1. Significant changes of transcript abundance in (A) shoots and (B) roots in response to treatments with aphids (A, blue), nematodes (N, yellow) or both herbivores (AN) in combination (red) in comparison to controls at 3 dpi and between the 3 treatments in (C) shoots and (D) roots of Arabidopsis thaliana analyzed with whole genome microarrays. Total number of genes with changed transcript levels and number of up- (") and downregulated (#) genes are indicated. The cut-off for differentially regulated genes was set to the adjusted P-value 0.05 (according to Benjamini and Hochberg25) and a log fold change of ¡0.5 (downregulation) or 0.5 (upregulation) (N D 3).
transcription occurred only in 40 genes (Fig. 1A and C). A and AN treatments mostly up-regulated the expression of a few genes with only some overlap between the effects caused by each treatment when compared to control plants (Fig. 1A). In contrast to our results, most other studies found more genes to be affected and mainly down-regulated by aphid feeding6,7,10,15, but see16. This can most likely be attributed to varying experimental conditions such as differences in induction duration, higher numbers of aphids used for infestation and analyses of other A. thaliana ecotypes or other aphid species.6,7,15 Nematode infestation caused no transcriptional changes in the systemic shoots in accordance with the lack in effects on metabolites and phytohormones reported earlier.1 When comparing the changes in shoot gene expression between the 3 herbivore treatments, no difference was
e976113-2
found between A and AN plants (Fig. 1C), but 37 genes were differentially regulated by A and AN compared to N plants with an overlap of 14 genes. Thus, although exclusive nematode infestation did not affect shoot gene expression, nematodes influenced the plant response to aboveground herbivory to some extent. Genes affected in the shoots were related to different levels of plant defense cascades, such as PR-protein coding genes, R-genes involved in gene-to-gene resistance, and genes related to oxidation-reduction processes (Table S1). Furthermore, genes involved in JA metabolism and signaling were induced, while SA-related genes seemed not to be affected in contrast to findings by Kusnierczyk et al.15 Moreover, genes relevant for primary and secondary metabolite biosynthesis, particularly of glucosinolates, and genes connected to myrosinase enzymes, were induced by A
Plant Signaling & Behavior
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and AN treatments. Expression of 2 of these genes (At2g24850, At1g52000) had been also found to be elevated in response to B. brassicae earlier7,10,15 while transcript levels of some of these genes were reduced by attack of a generalist aphid6 (Table S1). Interestingly, A and AN treatments also affected processes such as macromolecule organization, organonitrogen compound processes and proteolysis. Protein and amino acid degradation and nitrogen re-assimilation may be plant strategies to generate energy during aphid attack.10 Aphids can induce changes in the allocation of primary metabolites, particularly amino acids, for their own purpose.17,18 In the roots, only one gene (At5g05340), coding for a peroxidase, of the 90 genes affected by the herbivore treatments compared to the control was upregulated by the N treatment, which also responded to AN (Fig. 1B). This gene shows also increased expression in H. schachtii syncytia8 and in A. thaliana leaves following infestation with a generalist aphid6 (Table S2). The seemingly low responsiveness of root genes to nematode infestation may be due to a dilution effect when analyzing entire roots in contrast to the exclusive analysis of syncytia.8 In roots of younger plants, 128 genes with altered transcript levels caused by a more pronounced nematode infestation have been reported.5 Aphid feeding alone and in combination with nematodes evoked pronounced changes in gene expression in the roots compared to control plants, with distinct responses to A and AN infestation (Fig. 1B) as well as N versus AN (Fig. 1D). Our results support an earlier finding that B. brassicae alters root infestation rates by H. schachtii but not vice versa1,19, but see20,21. As in shoots, genes involved in diverse important stress responses such as oxidation-reduction processes, signaling events and several genes coding for transcription factors were up-regulated in the roots (Table S1). Furthermore, genes linked to cell References 1. Kutyniok M, M€ uller C. Crosstalk between above–and belowground herbivores is mediated by minute metabolic responses of the host Arabidopsis thaliana. J Exp Bot 2012; 63:6199-210; PMID: 23045608; http://dx. doi.org/10.1093/jxbers/274 2. Maffei ME, Mithofer A, Boland W. Before gene expression: early events in plant-insect interaction. Trends Plant Sci 2007; 12:310-6; PMID: 17596996; http://dx.doi.org/10.1016/j.tplants.2007.06.001 3. Sutter R, M€ uller C. Mining for treatment-specific and general changes in target compounds and metabolic fingerprints in response to herbivory and phytohormones in Plantago lanceolata. New Phytol 2011; 191:106982; PMID: 21592133; http://dx.doi.org/10.1111/ j.1469-8137.2011.03768.x 4. Soler R, Erb M, Kaplan I. Long distance root–shoot signalling in plant–insect community interactions. Trends Plant Sci 2013; 18:149-56; PMID: 22989699; http://dx.doi.org/10.1016/j.tplants.2012.08.010 5. Puthoff DP, Nettleton D, Rodermel SR, Baum TJ. Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant J 2003; 33:911-21; PMID: 12609032 6. de Vos M, van Oosten VR, van Poecke RM, van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ, Metraux JP, Van Loon LC, et al. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol Plant Microbe Interact 2005; 18:923-37; PMID: 16167763; http://dx.doi.org/10.1094/MPMI18-0923
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wall processes and secondary metabolite biosynthesis (glucosinolates, flavonoids, and one terpene biosynthesis gene) and degradation, potentially altering plant susceptibility to phytoparasitic nematodes,22,23 were influenced by A and AN treatments. Moreover, genes with responsiveness to water deprivation were affected, most likely because aphids withdraw high amounts of solutes and can induce osmotic stress in plants.10 Several of the genes regulated by AN treatment with importance for root or cell development, organization and growth were also differentially expressed in H. schachtii syncytia8 but some responded in an opposite direction. In summary, the effects on gene expression found here indicate that aphids alone and in combination with nematodes induce highly challenge-specific plant responses realized at diverse mechanistic defense levels even at low numbers of infestation. Aphids may affect systemic tissue due to strong sink capacities.24 Exclusive nematode infestation at an early time point of interaction with the host does not lead to relevant changes of gene expression but nematode presence alters the plant gene expression response to aphids especially in the roots. Future studies should address the question whether gene expression responses are enhanced when plants are attacked by higher infestation levels of aphids in combination with nematodes. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Supplemental Material
Supplemental data for this article can be accessed on the publisher’s website.
7. Kusnierczyk A, Winge P, Midelfart H, Armbruster WS, Rossiter JT, Bones AM. Transcriptional responses of Arabidopsis thaliana ecotypes with different glucosinolate profiles after attack by polyphagous Myzus persicae and oligophagous Brevicoryne brassicae. J Exp Bot 2007; 58:2537-52; PMID: 17545220 8. Szakasits D, Heinen P, Wieczorek K, Hofmann J, Wagner F, Kreil DP, Sykacek P, Grundler FM, Bohlmann H. The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant J 2009; 57:771-84; PMID: 18980640; http://dx.doi.org/10.1111/j.1365-313X.2008.03727.x 9. Kusnierczyk A, Tran DHT, Winge P, Jørstad TS, Reese JC, Troczy nska J, Bones AM. Testing the importance of jasmonate signalling in induction of plant defences upon cabbage aphid (Brevicoryne brassicae) attack. BMC Genomics 2011; 12:423; PMID: 21854623; http://dx.doi.org/10.1186/1471-2164-12423 10. Barah P, Winge P, Kusnierczyk A, Tran DH, Bones AM. Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection. PloS One 2013; 8:e58987; PMID: 23536844; http://dx.doi. org/10.1371/journal.pone.0058987 11. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of affymetrix GeneChip probe level data. Nucleic Acids Res 2003; 31:e15; PMID: 12582260 12. Lohse M, Nunes-Nesi A, Kr€uger P, Nagel A, Hannemann J, Giorgi FM, Childs L, Osorio S, Walther D, Selbig J, et al. Robin: an intuitive wizard application for R-based expression microarray quality assessment
Plant Signaling & Behavior
13.
14.
15.
16.
17.
18.
and analysis. Plant Physiol 2010; 153:642-51; PMID: 20388663; http://dx.doi.org/10.1104/pp.109.152553 R Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 2010 Thimm O, Bl€asing O, Gibon Y, Nagel A, Meyer S, Kr€uger P, Selbig J, M€ uller LA, Rhee SY, Stitt M. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 2004; 37:914-39; PMID: 14996223 Kusnierczyk A, Winge P, Jørstad TS, Troczy nska J, Rossiter JT, Bones AM. Towards global understanding of plant defence against aphids–timing and dynamics of early Arabidopsis defence responses to cabbage aphid (Brevicoryne brassicae) attack. Plant Cell Environ 2008; 31:1097-115; PMID: 18433442; http://dx.doi. org/10.1111/j.1365-3040.2008.01823.x Couldridge C, Newbury H, Ford-Lloyd B, Bale J, Pritchard J. Exploring plant responses to aphid feeding using a full Arabidopsis microarray reveals a small number of genes with significantly altered expression. Bull Entomol Res 2007; 97:523-32; PMID: 17916270 Sandstr€om J, Telang A, Moran N. Nutritional enhancement of host plants by aphids–a comparison of three aphid species on grasses. J Insect Physiol 2000; 46:33-40; PMID: 12770256; http://dx.doi.org/ 10.1016/S0022-1910(99)00098-0 Girousse C, Moulia B, Silk W, Bonnemain J-L. Aphid infestation causes different changes in carbon and nitrogen allocation in alfalfa stems as well as different
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21. Hol WHG, de Boer W, Termorshuizen AJ, Meyer KM, Schneider JH, van Der Putten WH, van Dam NM. Heterodera schachtii nematodes interfere with aphid-plant relations on Brassica oleracea. J Chem Ecol 2013; 39:1193-203; PMID: 24014097; http://dx.doi. org/10.1007/s10886-013-0338-4 22. Potter MJ, Vanstone VA, Davies KA, Kirkegaard JA, Rathjen AJ. Reduced susceptibility of Brassica napus to Pratylenchus neglectus in plants with elevated root levels of 2-phenylethyl glucosinolate. J Nematol 1999; 31:291-8; PMID: 19270899 23. Jones JT, Furlanetto C, Phillips MS. The role of flavonoids produced in response to cyst nematode infection
of Arabidopsis thaliana. Nematology 2007; 9:671-7; http://dx.doi.org/10.1163/156854107782024875 24. Kaplan I, Sardanelli S, Rehill BJ, Denno RF. Toward a mechanistic understanding of competition in vascularfeeding herbivores: an empirical test of the sink competition hypothesis. Oecologia 2011; 166:627-36; PMID: 21181415; http://dx.doi.org/10.1007/s00442010-1885-9 25. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B Met 1995; 57:289-300; http://www.jstor.orgstable2346101">http:www.jstor. orgstable2346101
Downloaded by [Washington University in St Louis] at 00:20 21 January 2015
inhibitions of longitudinal and radial expansion. Plant Physiol 2005; 137:1474-84; PMID: 15778456 19. Kutyniok M, M€ uller C. Plant-mediated interactions between shoot-feeding aphids and root-feeding nematodes depend on nitrate fertilization. Oecologia 2013; 173:1367-77; PMID: 23836091; http://dx.doi.org/ 10.1007/s00442-013-2712-x 20. Hol WHG, de Boer W, Termorshuizen AJ, Meyer KM, Schneider JHM, van Dam NM, van Veen JA, van der Putten WH. Reduction of rare soil microbes modifies plant-herbivore interactions. Ecol Lett 2010; 13:292-301; PMID: 20070364; http://dx.doi.org/ 10.1111/j.1461-0248.2009.01424.x
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Plant Signaling & Behavior
Volume 9 Issue 11
