Insect Science (2014) 21, 727–740, DOI 10.1111/1744-7917.12072

ORIGINAL ARTICLE

Response of Solanum tuberosum to Myzus persicae infestation at different stages of foliage maturity Adriana E. Alvarez1,2 , Anah´ı M. Alberti D’Amato2 , W. Fred Tjallingii1,3 , Marcel Dicke1 and Ben Vosman2 1 Laboratory 3 EPG

of Entomology, Wageningen University, 6700 EH Wageningen, 2 Plant Breeding, Wageningen UR, 6700 AA Wageningen, and systems, 6703CJ Wageningen, the Netherlands

Abstract Young leaves of the potato Solanum tuberosum L. cultivar Kardal contain resistance factors to the green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) and normal probing behavior is impeded. However, M. persicae can survive and reproduce on mature and senescent leaves of the cv. Kardal plant without problems. We compared the settling of M. persicae on young and old leaves and analyzed the impact of aphids settling on the plant in terms of gene expression. Settling, as measured by aphid numbers staying on young or old leaves, showed that after 21 h significantly fewer aphids were found on the young leaves. At earlier time points there were no difference between young and old leaves, suggesting that the young leaf resistance factors are not located at the surface level but deeper in the tissue. Gene expression was measured in plants at 96 h postinfestation, which is at a late stage in the interaction and in compatible interactions this is long enough for host plant acceptance to occur. In old leaves of cv. Kardal (compatible interaction), M. persicae infestation elicited a higher number of differentially regulated genes than in young leaves. The plant response to aphid infestation included a larger number of genes induced than repressed, and the proportion of induced versus repressed genes was larger in young than in old leaves. Several genes changing expression seem to be involved in changing the metabolic state of the leaf from source to sink. Key words cDNA microarrays, green peach aphid, insect–plant interactions, potato

Introduction Aphids are phloem feeding insects that closely interact with their host plant. On suitable host plants, the aphids will choose the best feeding place and different aphid species on a same plant often have different preferences.

Correspondence: Ben Vosman, Plant Breeding, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, the Netherlands. Tel: +31 317 480838; fax: +31 317 483457; email: [email protected] Present address: Adriana E. Alvarez and AnahI´ M. Alberti D Amato, Facultad de Ciencias Naturales, Universidad Nacional de Salta, Av. Bolivia 5150, 4400-Salta, Argentina.

Brevicoryne brassicae usually occurs on the youngest leaves of growing cabbage plants, while Myzus persicae is more likely to be found on the oldest, senescing leaves of cabbage (Blackman, 1974). On potato plants, leaves of different maturity stages offer different environments to aphids. For example, M. persicae prefers to settle on the potato Solanum tuberosum L. cultivar Kardal underneath older leaves at the time the leaves turn yellow. The aphids reproduce, accumulate against the midrib and secondary veins, and move upward progressively to the next leaf to become yellow soon, while the apical young leaves remain free of aphids. Previous studies on probing behavior and colony development on cv. Kardal showed a certain degree of resistance to M. persicae present in young apical leaves that decreased as soon as the leaves

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became older (Alvarez et al., 2006, 2007). The physiological mechanism that confers resistance to young upper leaves is at present unknown. Resistance to pathogens, like the oomycete Phytophthora infestans (Mont.), seems to follow a similar pattern. In resistance to late blight the leaf position is a significant factor since apical leaves are far more resistant than basal leaves (Visker et al., 2003). On potato plants the M. persicae reproduction rate tends to be greater on senescing leaves and differences in chemical composition of the leaves have been suggested to drive the aphid preference for senescence leaves (Aldamen & Gerowitt, 2009). Also on potato plants with induced senescence the aphid performance and feeding behavior of M. persicae is enhanced through an increase in sap ingestion that likely resulted in a reduction in developmental time and improved plant acceptance by M. persicae (Machado Assefh et al., 2013). In tobacco plants, there is a change in the composition of the phloem sap when leaves senesce, that is, carbohydrates decrease and total amino acids increase (Masclaux-Daubresse et al., 2000; Masclaux-Daubresse et al., 2006). Several studies have shown that differences in geneexpression occur during senescence of leaves and that those changes indicate changes in the physiology of the leaf. Senescence is an active developmental and highly regulated process that includes the modulated expression of many genes related to different functional categories (Buchanan-Wollaston et al., 2003, 2005; Gepstein et al., 2003). The degradation of chloroplasts is one of the key factors in leaf senescence, resulting in recycling and mobilizing the nutrients from senescing leaves to developing sink parts (Buchanan-Wollaston, 1997). Chlorophyll loss is usually accompanied by the release and breakdown by ribulose biphosphate carboxylase oxygenase (RuBisCO), which comprises more than 50% of the protein in green leaves (Wittenbach, 1979). The resulting mobilization of amino acids enriches the phloem sap and may stimulate phloem sap ingestion by aphids. Phloem feeding by aphids is hydraulically equivalent to natural plant sinks, such as fruits or roots. However, in susceptible plant–aphid relationships there are more complex interactions than between natural source–sink tissues in plants (Douglas, 2003). There is evidence that aphids influence source– sink relationships. In Nicotiana attenuata some genes that are constitutively differentially expressed in source or in sink leaves, change their expression after the attack of Myzus nicotianae (Voelckel et al., 2004). In some plant–aphid interactions, induced senescence is related to plant acceptance by the aphid (Al Mousawi et al., 1983; Morgham et al., 1994; Machado Assefh et al., 2013). Furthermore, the altered carbohydrate mobilization from aphid-injured leaves might be the trig C 2013

ger for physiological events with secondary effects such as reduction in photosynthesis and chloroplast degradation (Franzen et al., 2008). Studies on wheat (Franzen et al., 2007) and barley (Gutsche et al., 2009) infested with the Russian wheat aphid (Diuraphis noxia Mordvilko) and soybean infested with soybean aphid (Aphis glycines Matsumura) (Pierson et al., 2010) have shown that resistant genotypes endure aphid feeding by maintaining their photosynthesis rate. Susceptible plants infested by aphids show a fast decline in photosynthesis. Hence in susceptible plants, aphid feeding has a negative effect on the carbon-linked/dark reaction of photosynthesis, that is, rubisco activity and regeneration. But until now little is known about the mechanisms and genetic basis of increased susceptibility of senescent leaves to aphids. One hypothesis is that on susceptible plants, aphids induce senescence-like changes to increase breakdown and translocation of leaf proteins (Dorschner et al., 1987). The drastic increase of glutamine in phloem exudates after aphid feeding supports this hypothesis (Sandstr¨om et al., 2000). Glutamine is considered the major compound by which nitrogen translocation from senescent leaves to sink organs in rice and other plants takes place (Kamachi et al., 1992; Watanabe et al., 1997; MasclauxDaubresse et al., 2008). Here, we compared the settling by M. persicae on young and old leaves of S. tuberosum cv. Kardal, and specifically address the differences in gene expression between young (incompatible interaction) and mature leaves (compatible interaction) of cv. Kardal in response to M. persicae probing and feeding.

Materials and methods Plants and aphids Solanum tuberosum L. cultivar Kardal was selected for this study because of its high level of resistance to M. persicae in apical leaves. This resistance decreases in mature and senescent leaves (Alvarez et al., 2006). The plants were maintained and propagated in vitro on Murashige and Skoog medium including vitamins, sucrose 3%, pH 5.8. After 2 weeks on agar the plantlets with developed roots were transferred to soil in 22-cm diameter pots in a glasshouse at 22 ± 2 ◦ C, 70% RH, and 16 : 8 h light : dark photoperiod. Aphids used in the experiments came from newly established clones from a single virginoparous apterous individual taken from an M. persicae colony maintained at the Laboratory of Entomology, Wageningen University. Aphids were reared on radish, Raphanus sativus L., in Institute of Zoology, Chinese Academy of Sciences, 21, 727–740

Response of young and old potato leaves to aphids

cages in a climate chamber at 22 ± 2 ◦ C, 30%–40% RH, and 16 : 8 h light : dark photoperiod. New synchronous colonies were started weekly from which newly molted adults were used to perform infestations.

Aphid settling behavior test To study differences in the settling by M. persicae on young versus old leaves 20 aphids were put on plants (time 0), 10 on each of 2 test leaves and using 6–8 plant replicates per treatment. In order to prevent aphids from moving to other leaves, nontest leaves were entirely enveloped with nonwoven bags, leaving only the test leaves and the stems accessible to the aphid’s probing and walking. To avoid aphid migration from one plant to the other, the plants were individually surrounded by water in a tray. Aphids that would move away from the plant drowned in the water or kept walking around the pot. The number of aphids remaining on the test leaves after 30 min, 16, 21, and 38 h was counted. The numbers of aphids settling on young or old leaves over time were processed by regression analysis. The Student’s t analysis was used to test the regression coefficient, for linear relationship between numbers of aphids and time. Means and standard error of means (SEM) of the numbers of aphids per plant at each time were calculated. The Mann–Whitney U test was used to analyze the data on aphid settling behavior for young and old leaves at each time point.

Plant infestation with aphids To study the transcriptomic response of cv. Kardal to M. persicae, we infested 45–55-d-old plants with aphids. Plants were arranged in a glasshouse at 22 ± 2 ◦ C, 70% RH, and 16 : 8 h light : dark photoperiod in a randomized complete design. Infestation was carried out either on young or on old leaves. Plants were assigned to 4 treatments: A (young leaves infested), B (control young leaves noninfested), C (old leaves infested), and D (control old leaves noninfested). The young leaves used were numbers 2 and 3 from the apex while mature-old leaves were numbers 7–9 leaves from the apex. Two young or 2 old leaves per plant were preinfested with 40 young adult apterous aphids per leaf. Aphids were confined to the leaves by enveloping each leaf individually with nonwoven “agrotextile” bags and plants were placed on individual trays. Leaves of control plants were also enveloped with nonwoven bags without adding aphids. Infestation went on for  C 2013

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96 h, after that aphids and nymphs were carefully removed with a soft brush. Sample preparation and cDNA microarray hybridizations The leaves were cut from the plant, weighed, immediately frozen in liquid nitrogen, and stored at −80 ◦ C until use for RNA isolation. Complete leaves were taken from 4 plants per treatment for analysis of gene-expression in young and old leaves. RNA was isolated from leaves of 3 plants (biological replicates), while the leaves from the 4th plant were kept as a backup. The 2 infested leaves of each plant were pooled. Total RNA was extracted from frozen leaves with TRIzol (Invitrogen, Life Technologies Corporation, Grand Island, NY, USA) and purified using the RNeasy mini elute kit (Qiagen, Valencia, CA, USA). cDNA was obtained from total RNA by Super Script II reverse transcriptase (Invitrogen) with POLYdT primers and was purified with QIAquick PCR purification kit (Qiagen). The cDNA was labeled with cyanine 3 (Cy3), and cyanine 5 (Cy5) fluorescent dyes (Amersham). cDNA was prepared and labeled as previously described (Alvarez et al., 2013). The array used for the hybridization contains a collection of 3 570 S. tuberosum cDNA clones (Alvarez et al., 2013). To analyze differential gene expression, RNA from infested leaves was mixed with RNA of noninfested control leaves, generating 2 combinations: (i) M. persicae infested young leaves versus the noninfested control young leaves (at the same 2–3 leaf position), and (ii) M. persicae infested old leaves versus noninfested control old leaves (at the same 7–9 leaf position). Three slides were hybridized for each treatment using cDNA of different plants (3 biological replicates). Micro-array data analysis Signal and background fluorescence intensities of the arrays were analyzed using the ScanArray Express program (Perkin-Elmer) version 2.22. Arrays were checked manually to exclude anomalous spots with high background from the analysis. Spots with fluorescence intensities lower than half the background were raised to half the background to avoid extreme expression ratios; when both dyes had intensities lower than half the background, they were excluded. Data were converted by Express Converter version 1.5, log 2 ratios of Cy5/Cy3 were calculated and normalized to avoid spatial bias within each slide using the Locfit (Lowess) normalization method by the TIGR Microarrays Data Analysis System (MIDAS),

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and old leaves (Mann–Whitney U test, at 30 min: U = 55.5, P = 0.7290, at 16 h: U = 18, P = 0.1980), after 21 h significantly fewer aphids were recorded on young than on old leaves (Mann–Whitney U test, U = 99.5, P = 0.0003). Finally, after 38 h, only 10% of the initial aphids were found on the young leaves, compared to 52% of aphids found on the old leaves (Mann–Whitney U test, U = 84, P = 0.0003) (Fig. 1).

Number aphids Numberof of aphids perper plantplant

20

15

10

y = −0.0488x + 12.807 R² = 0.0469

5 y = −0.2521x + 11.926 R² = 0.6168

0

0

5

10

15

25 20 Time Time (h)(h)

30

35

40

Fig. 1 Settling behavior of Myzus persicae on Solanum tuberosum cv. Kardal leaves of different age. At the start of the experiment all plants had 20 aphids each, then the number of aphids remaining on the plants after 0.5, 16, 21, and 38 h was counted. Regression lines are shown for settling of aphids on young leaves () overtime (t = −6.34, P = 0.0001), and old leaves () overtime (t = −1.11, P = 0.2781), respectively. There is a difference of aphid settling between young and old leaves maturity stage at 21 and 38 h (Mann–Whitney U test, U = 99.5, U = 84, P = 0.0003, respectively).

Version 2.19. TIGR MEV version 3.0.3 was used to perform t-test statistical analysis on the log 2 ratios; genes with expression ratio 2 folds higher (log 2 ratios ≥ 1) were consider upregulated, and genes with expression ratio 2 folds lower (log 2 ratios ≤ −1) were considered downregulated, when statistically different from 0 (P ≤ 0.05). We also included some genes clearly upregulated in all 3 biological replicates with an expression log 2 ratio ≥ 1 but with P value between 0.05 and 0.1 due to a high variability between replicates. The criterion to be included was that at least 2 of 3 technical replicates on each slide showed differential regulation. The absolute number of differentially expressed genes on young and old Kardal leaves was compared by chi-square test. Results Settling behavior of M. persicae on S. tuberosum cv. Kardal The results on the settling behavior of aphids on young leaves showed a significant decrease in the number of aphids remaining on the leaves over time (R = −0.785, t = −6.34, P = 0.0001). In contrast, on old leaves, no decrease in the number of aphids over time was observed (R = −0.214, t = −1.11, P = 0.2781) (Fig. 1). While at 30 min and 16 h after the start of the experiment there was a similar number of aphids settled on both young  C 2013

Gene expression in response to aphid feeding Transcriptional responses of cv. Kardal plants to M. persicae infestation in young and old leaves were compared. In old leaves, M. persicae infestation elicited a stronger response than in young leaves; 95 genes (61 upregulated and 34 downregulated) versus 55 genes (48 upregulated and 7 downregulated) were differentially expressed, respectively (Fig. 2A, 2B) (χ 2 value = 9.3, df = 1, P = 0.0023). The proportion of downregulated genes relative to the total number of genes with changed expression was 36% (34/95) in old leaves, and 13% (7/55) in young leaves, therefore in old leaves there is a 2.8 folds higher number of genes repressed than in young leaves. The differentially regulated genes are listed in Table 1. Genes have been assigned to functional category based on their annotation on TIGR Plant Transcript Assemblies database (http://plantta.jcvi.org/). This grouping confers a potential function to each encoded protein based on similarity to proteins with known functions (Fig. 2C, 2D). Most of the downregulated genes were found in old leaves. The number of downregulated genes in young leaves of cv. Kardal was small (Fig. 2, Table 1). Discussion Settling behavior of M. persicae Aphid settling on young leaves did not significantly differ from old leaves during the 1st 16 h of the experiment. However, a considerable reduction in the number of aphids settling on young leaves is seen 21 h after the start of the experiment (Fig. 1). This suggests that the aphids first probe the plant and leave it sometime afterwards when encountering feeding constraints. Hence, the plant resistance is not located at surface level, that is, at the cuticle–epidermis of young cv. Kardal leaves, but deeper in the plant tissue and likely phloem-based because the aphids leave the plant after several hours of probing. These results are in line with those on the probing behavior of aphids using the electrical penetration graph (EPG) (Alvarez et al., 2006; Tjallingii, 2006). On young leaves Institute of Zoology, Chinese Academy of Sciences, 21, 727–740

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Fig. 2 Genes differentially expressed in old and young leaves of Solanum tuberosum cv. Kardal infested after 96 h of attack by Myzus persicae. Venn diagrams showing differences in the number of genes up (A) or down (B) regulated after infestation. Differences in the number of genes up (C) or down (D) regulated across gene-functional categories.

of cv. Kardal, the aphids take the same amount of time to the 1st salivation into the phloem as on susceptible cultivars, whereas phloem sap ingestion takes significantly longer on cv. Kardal than on the susceptible potato plants (Alvarez et al., 2006), suggesting that young leaves of cv. Kardal have resistance factors at the phloem level that inhibit colonization of young leaves. Gene expression in young and old leaves of cv. Kardal We found 104 differentially regulated genes in cv. Kardal after 96 h of M. persicae infestation, thus only 3% of the genes present on the array responded. This is comparable to the number of genes responding in S. stoloniferum to M. persicae or M. euphorbiae attack (4%) using the same cDNA microarray (Alvarez et al., 2013). It should be noted that these genes represent only a subset of the total number of genes responding to aphid attack, because we use a dedicated cDNA microarray on which mainly genes involved in plant responses to pathogens were spotted. In old leaves of cv. Kardal infested by M. persicae as well as in S. stoloniferum leaves infested by M. euphor-

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biae (Alvarez et al., 2013) the number of genes that change expression in the compatible interaction is larger than the number of genes that change expression in the incompatible interactions, i.e., young leaves cv. Kardal infested by M. persicae, and S. stoloniferum infested by M. persicae. It is important to realize that we measured gene expression in plants at 96 h postinfestation, thus in the compatible interaction and according to the criterion of “sieve element acceptance” by the aphid (Tjallingii & Hogen Esch, 1992), this period is long enough for host plant acceptance to occur (Alvarez et al., 2006). Therefore, at this time the expression of genes related to stress response, to transport of molecules, and to compensate for the withdrawal of nutrients will be differentially regulated. On the other hand, at 96 h postinfestation the changes in gene expression occurring in the early stages in the plant–aphid interaction will have vanished, for example, early genes related to signaling and oxidative stress (Maffei et al., 2007; Kusnierczyk et al., 2008). Overall we found that in the plant response to aphid attack, a larger number of genes were induced than repressed (Fig. 2). Other authors also found a larger number of induced than repressed genes (Couldridge et al., 2007; Kusnierczyk et al., 2008; Alvarez et al., 2013).

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General metabolism Protein metabolism

Regulatory

PR

Protein metabolism Lipid metabolism

Regulatory

PPCCF17 PPCBF82

PR

PPCAH09

STMJF47 STMDF49 cSTS18G22

BPLI1H2 POAE058

STMEY20

STMJC14 cSTB33M4

PPCBM08 PPCAI63 PPCCM26 BPLI1G3 PPCBJ33 PPCAS26 PPCAH78

PPCAC06 BPLI4D2 PPCAT70

PPCCP54 PPCBP46 PPCAT19

Clone name

Functional category PR protein STH-21, Solanum tuberosum (100%) PR-1 protein isoform b1, S. tuberosum, Solanum lycopersicum (100%) PR-2 protein P2, S. lycopersicum (100%) PR-2 protein 1,3-β-glucanase, S. tuberosum (37%) Endo-1,3-β-glucosidase acidic isoform GI9, Nicotiana tabacum (85%) Class II (acidic) chitinase, S. tuberosum (100%) Class IV chitinase, A. thaliana (91%) PR protein R major form (Thaumatin-likeprotein E22), N. tabacum (100%) WRKY-DNA-binding protein 4, N. tabacum (66%) LRR protein (CALRR1), N. tabacum (22%) Auxin-induced/SAUR-like protein, C. annum (100%) Protein kinase like protein, A. thaliana (38%) Isoleucyl-tRNA synthetase, A. thaliana (5%) Nonspecific lipid transfer protein, S. tuberosum (83%) Xylogen protein 1/nonspecific lipid transfer protein, A. thaliana (55%) Unknown protein hsr203J, N. tabacum (100%); NgCDM1, N. glutinosa (100%) Endochitinase (Chitinase), S. tuberosum, S. lycopersicum (100%) NPR1-interactor protein 1, S. lycopersicum (94%) AP2 domain-containing transcription factor TINY, A. thaliana (6%) S-receptor kinase (SRK), A. thaliana (31%) AAA-type ATPase family protein, A. thaliana (48%) Chloroplast nucleoid DNA binding prot. 41 kD (CND41), N. tabacum (92%) PR subtilisin-serine-like protease (P69B), S. lycopersicum (46%)

Gene annotation (putative function), degree homology with other spp (%) Young 3.57** 3.80** 4.83* 3.05* 2.49** 2.34* 2.30** 4.96* 3.13** 2.57** 2.58* 2.89** 4.35* 3.13** 3.97* 3.84** 2.19** 2.03** 2.13** 2.22** 3.45** 2.47** 2.47** 3.00**

Old† 3.24** 3.76** 3.91* 4.19* 2.71** 2.74* 2.52** 4.60* 2.19** 3.10** 4.63* 2.58** 3.81* 2.53** 3.76* 3.06** 2.73** 3.07** 2.14** 2.80** 3.37** 3.57** 3.42** 5.13**

Protein catabolism

Signalling Variety of cell process Rubisco catabolism

Regulation transcription Regulation transcription

Defence/antifungal

Unknown Defence/cell death

Regulation transcription Regulation Signalling Signalling Protein translation Lipids transport Cell differentiation

Defence/antifungal Defence/antifungal Defence/antifungal

Defence related Defence/antifungal Defence/antifungal

Defence related Defence related

Process category

Table 1 Genes with changed expression in Solanum tuberosum cv. Kardal old and young leaves after infestation for 96 h with Myzus persicae.

(to be continued)

P/ET/SA

S

SA AS

P

P/TMV

P/SA P/AS/W/ABA P

P/SA P P/ET

P P/SA P/SA

P/W P/SA

Response to‡

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PPCBN57

Regulatory Intracell transport

PR

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BPLI3K16

POABC66 PPCCF11 PPCBL95

STMHZ79

Protein metabolism

Transport Cell death inhibitors

PR

BPLI4I8

PPCBH33 PPCCI93

Regulatory

POADE25

PPCAC11 POABE75

POAEE44

PPCAI46

PPCBL51 PPCAI68 POCBT09 PPCBI30 PPCAR90 PPCCL54 POCBT23

Transport

Unknown

Clone name

Functional category

Table 1 Continued.

Amino acid transport protein AAT1, A. thaliana (65%) Hexose transporter, S. lycopersicum (39%) Hexose transporter, S. lycopersicum (83%) Unknown protein, N. tabacum (60%) Unknown protein Nucleic acid binding protein, A. thaliana (47%) Coatomer beta’ subunit (Beta’-coat protein), A. thaliana (28%) PR-1 prot. (Prb-1b), N. tabacum (94%); Basic PR-1 precursor, C. annum (91%) Endochitinase (Chitinase), S. tuberosum, S. lycopersicum (100%) PR-5 prot., S. lycopersicum (92%); Osmotin-like prot., N. tabacum (95%) Osmotin-like protein OSML13 (PA13), C. annum (100%) Osmotin-like protein OSML15 (PA15), S. commersonii, C. annum (100%) Osmotin-like protein OSML81 (PA81), S. commersonii, C. annum (100%) Zinc knuckle (CCHC-type) family protein, A. thaliana (9%) Lectin (probable mannose binding) protein kinase, A. thaliana (27%) Multifunctional aminoacyl-tRNA ligase-like protein, A. thaliana (92%) Ring finger protein, Cicerarietinum (65%) NtEIG-A1 protein, N. tabacum (76%) MLO-like protein 12 (AtMlo12) (AtMlo18), A. thaliana (22%) Disease resistance-responsive prot., dirigent prot., A. thaliana (47%) Disease resistance protein Hcr2–5D (LRR), S. lycopersicum (21%)

Gene annotation (putative function), degree homology with other spp (%) Young 2.18** 4.15** 2.26** 2.24** 2.03** 4.19** 2.42** 2.64** 2.02** 3.14** 3.38** 3.22** 3.41** 2.24** 2.26** 2.03** 2.01** 3.74** 2.32** 1.75 2.21

Old† 2.32** 4.72** 2.74** 2.63** 2.22** 3.19** 2.41** 3.41** 2.37** 3.21** 2.68** 2.89** 2.75** 2.62** 2.82** 2.00** 2.03** 4.26** 2.31** 2.63** 3.71**

Signaling

Defense related

Protein ubiquitination Electron transport Cell death inhibitors

Protein translation

Regulation transcription Signalling

Defence/antifungal

Defence/antifungal Defence/antifungal

Defence/antifungal

Defence/antifungal

Defence related

Amino acid transport Carbohydrate transport Carbohydrate transport Unknown Unknown Regulation transcription Protein transport

Process category

(to be continued)

P

P

P

P/AS/W/ABA/SA

P/AS/W/ABA/SA P/AS/W/ABA/SA

P/AS

P

P/AS/ET

TMV/SA

Response to‡

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PPCBD19

Regulatory

PPCBB86 PPCAQ66

Unknown

Regulatory PR

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cSTA43M16

cSTE21I23

Dormancy-related

Regulatory

Regulatory Lipid metabolism Unknown

Protein metabolism

BPLI7M2 PPCAU07 PPCBB93

PPCAQ85 PPCBH37 STMJE25 PPCBG74 PPCAN32 PPCAC18 PPCBD84 PPCCK15

Regulatory Unknown

PPCAW92 STMDK87

Protein metabolism

STMJD51

STMHQ95 STMJE78 PPCAR85

PPCBM14

Clone name

Functional category

Table 1 Continued.

Receptor-like serine-threonine protein kinase, S. tuberosum (31%) Strubbelig receptor family 1–LRR protein kinase, A. thaliana (35%) Calmodulin-binding family protein, A. thaliana (39%) Silencing group B protein, Zea mays (100%) Branched-chain amino acid aminotransferase, S. tuberosum (38%) Ubiquitin-specific protease 12 (UBP12), A. thaliana (11%) KED, lysine rich (K), glutamic (E) and aspartic acid (D), N. tabacum (38%) Wall-associated kinase 1 (WAK1), A. thaliana (27%) Pathogenesis related protein, Oryzasativa (43%), Hordeum vulgare (39%) Disease resistance protein Hcr9–9E, S. pimpinellifolium (22%) Basic PR-1 protein, C. annuum (100%) Rxprotein (LZ-NBS-LRR protein), S. tuberosum (96%) Endo-1,3-β-glucosidase, basicisoform 2 precursor, S. tuberosum (100%) Eukaryotic protein kinase domain, A. thaliana (30%) Unknown protein, A. thaliana (89%) Unknown protein VQ motif putative, O. sativa (17%) Enoyl CoA hydratase, A. thaliana (93%) Unknown, A. thaliana (96%) Unknown Protein-methionine-S-oxidereductase, S. lycopersicum (100%) Dormancy/auxin-repressed protein, Solanum virginianum (100%) Ethylene-responsive element binding factor, N. tabacum (85%)

Gene annotation (putative function), degree homology with other spp (%)

1.32 1.50 1.87 1.44 1.27 2.30 1.40 2.69

2.72** 2.22** 2.91** 2.06** 2.01** 7.30** 1.94** 3.78**

1.04 1.93 1.81 1.47 1.48 1.54 2.65** 2.28** 2.49** 1.99** 3.12** 0.43** 0.38**

3.72** 2.31** 2.73** 2.76** 2.08** 2.03** 2.12 1.78 1.93 1.47 1.3 0.42** 0.18**

1.33

1.73

2.72**

2.58**

Young

Old†

Regulation transcription

Dormancy

Signaling Unknown Unknown Regulatory Fatty acid metabolism Unknown Unknown Protein catabolism

Defense related Defense related Defense/antifungal

Defense related

Signaling Defense related

Protein ubiquitination Unknown

Signaling Regulation growth Aminoacid metabolism

Signaling

Signaling

Process category

(to be continued)

AS/ET

P P/PVX P/W/ET

P

P P

W

P

Response to‡

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PPCBZ61 PPCBN68 BPLI3F3 STMJG06 POCCL34 cSTE16K18

Protein metabolism Regulatory General metabolism PR

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cSTB24E4 cSTD1B23

cSTD3N22

cSTB47G10

cSTD7D10 cSTB40I24 cSTB25F9 cSTB45H11

cSTB11M8

Protein metabolism

Lipid metabolism

Second metabolism

Cell-wall metabolism

cSTB36A4 cSTD18K14 cSTD17G10

General metabolism General metabolism

Regulatory

cSTD2E22

Lipid metabolism

PPCAE52 POACZ85 cSTB43O7 POAB559

Clone name

Functional category

Table 1 Continued.

Patatin T5 precursor (Potato tuber protein), S. tuberosum (100%) Zinc finger (C3HC4-type RING finger), A. thaliana (56%) Protein phosphatase 2C putative (PP2C), A. thaliana (69%) Aldo/ketoreductase, A. thaliana (87%) PR protein, A. thaliana (61%) PR Thaumatin-like protein, A. thaliana (68%) Major latex like protein homolog, Beta vulgaris (70%), A. thaliana (46%) Wound stimulated protein Sn-1 protein, C. annum (52%) Speckle-type BTB/POZ, A. thaliana (62%) MYB transcription factor, A. thaliana (49%) GIA/RGA-gibberellin response modulator, A. thaliana (70%) CDC5 protein, Z. mays (57%) Zinc finger protein 4, O. sativa (29%); A. thaliana (21%) Zinc-finger protein Stress-associated protein-3 (SAP-3), O. sativa (73%) Glycosyltransferase family 8 protein, A. thaliana (93%) Anthocyanidin 3-O-glucosyltransferase, Petunia hybrida (96%) Type I small heat shock protein 17.6 kDa isoform, S. lycopersicum (100%) Nonspecific lipid-transfer protein 1 (LTP 1), S. tuberosum (94%) Chalcone-flavononeisomerase B, P. hybrida (95%) Naringenin-chalcone synthase 2, S. tuberosum (100%) Naringenin-chalcone synthase 1A, S. tuberosum (100%) Proteinase inhibitor type II TR8 precursor, S. lycopersicum (44%) CDH1-D gene for 18S rRNA, Extensin-like S. tuberosum (100%)

Gene annotation (putative function), degree homology with other spp (%) Young 0.25* 0.52 0.67 0.64 0.73 0.61 0.94 0.53 0.66 0.55 0.73 0.78 0.99 0.55 0.93 0.80 0.94 0.73 0.55 0.46 0.76 0.71 0.71

Old† 0.07** 0.38** 0.47** 0.40** 0.42** 0.40** 0.48** 0.31** 0.37** 0.43** 0.47** 0.34** 0.49** 0.40** 0.34** 0.29** 0.44** 0.47** 0.24** 0.30** 0.45** 0.20** 0.31**

Cell-wall biogenesis

Flavonoid biosynthesis Flavonoid biosynthesis Flavonoid biosynthesis Secondary metabolism

Lipids transport

Protein folding

Carbohydrate synthesis Metabolism

Regulation transcription Regulation transcription Regulation transcription

Defense related Unknown Regulation transcription Regulation transcription

Protein ubiquitination Regulatory Metabolism Defense related Defense/antifungal Unknown

Nutrient reservoir

Process category

(to be continued)

P/W

P P P

Response to‡

Response of young and old potato leaves to aphids 735

PPCAW72

Transport

PPCAD09

Photosynthesis

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cSTE17A9 PPCBB35

PPCBB22

Protein metabolism

Intracell transport Transport

Unknown

Aquaporin 1, N. tabacum (100%); Tonoplast intrinsic prot., Zea mays (98%) Proton-dependent oligopeptide transport (POT) protein, A. thaliana (11%) Copper chaperone-farnesylated protein ATFP6, A. thaliana (88%) Ntdin-Senescence-associated protein DIN1, N. tabacum (83%) Thylakoid lumenal 19 kDa protein, chloroplastic, A. thaliana (81%) Unknown protein, O. sativa (43%) Unknown protein, O. sativa (32%) Unknown protein Unknown protein SKP1-like prot-E3 ubiquitin ligase SFC complex, N. benthamiana (100%) Hypothetical protein, C. arietinum (72%), O. sativa (47%) Sodium sulfate or dicarboxylate transporter, A. thaliana (83%) Unknown protein, A. thaliana (37%)

Gene annotation (putative function), degree homology with other spp (%)

Protein transport Transport/cell maintenance Unknown

0.82 0.63 0.73 0.68 0.51 0.48 0.56 0.48** 0.45** 0.50** 0.49**

0.41** 0.34** 0.43** 0.42** 0.45** 0.40** 0.28** 0.42

0.49

0.40 0.63

Unknown Unknown Unknown Unknown Protein catabolism

0.59

0.41**

Calcium ion binding

Aging/senescence

Metal-ion transport

Oligopeptides transport

Water transport

0.53

0.32**

Process category

Young

Old†

Response to‡

Values were calculated as relative transcript abundance (ratios of values for aphid infested plants/values for control plants). The symbol ** indicates genes with expression ratio 2 folds higher (upregulated) or 2 folds lower (downregulated) than the control (P ≤ 0.05). The symbol * indicates genes upregulated in all 3 biological replicates with expression ratio 2 folds higher or 2 folds lower but with 0.05 ≤ P ≤ 0.1 due to a high variability between replicates. The criterion to be included was that at least 2 of 3 technical replicates in the slide showed differential regulation; Old, fold change in Kardal old leaves after M. persicae attack; Young, fold change in Kardal young leaves after M. persicae attack. ‡ P, pathogens; W, wounding; AS, abiotic stress; S, senescence; SA, salicylic acid; ET, ethylene; JA, jasmonic acid; ABA, absicic acid; TMV, tobacco mosaic virus; PVX, potato virus X.

†

cSTB6I18 cSTB1M4 PPCAY05 cSTB29G22 cSTB43B10

Unknown

cSTB48I2

Aging/senescence

cSTB49A23

BPLI3P23

Clone name

Functional category

Table 1 Continued.

736 A. E. Alvarez et al.

Institute of Zoology, Chinese Academy of Sciences, 21, 727–740

Response of young and old potato leaves to aphids

Interestingly, while many of the upregulated genes were the same in old and young leaves, the downregulated genes differed largely between the 2 leaf types (Fig. 2). The results obtained here for cv. Kardal showed overlap with the results obtained previously for S. stoloniferum upon M. persicae or M. euphorbiae attack (Alvarez et al., 2013). Solanum stoloniferum and S. tuberosum cv. Kardal had a larger overlap among the induced genes than that among the repressed genes (results not shown). In contrast, the responses of 2 Brassica oleracea cultivars to infestation by the aphid B. brassicae were cultivar specific. In that response, there was only one induced and one repressed gene in common between both cultivars (Broekgaarden et al., 2008). Upregulated genes The gene-expression studies provided indirect evidence that at a late time–after 96 h–postaphid infestation of cv. Kardal plants respond by activating genes of the SA and ethylene (ET) signal transduction pathways. The attack of M. persicae on cv. Kardal did not lead to the upregulation of any jasmonic acid (JA) responsive gene although there were many JA responsive genes on the cDNA array. However, it should be noted that in Arabidopsis thaliana infested by B. brassicae, JA responsive genes were activated early in the interaction and that their expression dropped at 12 h postinfestation (Kusnierczyk et al., 2008); nevertheless on cv. Kardal at 96 h the SA signaling transduction pathway prevailed. In old and young leaves of cv. Kardal M. persicae induced the expression of a number of PR genes. PR genes have been associated with aphid feeding in diverse plant– aphid interactions (Moran & Thompson, 2001; Moran et al., 2002; Zhu-Salzman et al., 2004; De Vos et al., 2005; Park et al., 2006), and most of these genes are responsive to salicylic acid (SA), suggesting that the SA signaling pathway is still activated 96 h postaphid infestation. These results are in agreement with the “decoy” hypothesis that proposes aphids to manipulate plant defense responses, by inducing the SA-signaling pathway to suppress the biologically more effective JA-signaling pathway (Zhu-Salzman et al., 2004; Thompson & Goggin, 2006). Like in both S. stoloniferum–aphid interactions (Alvarez et al., 2013), we also found in young and old leaves of cv. Kardal upregulated genes that could play a role in switching the tissue status from source to sink. One is the xylogen protein 1/nonspecific lipid transfer protein (nsLTP), which occurs in sink tissues (Motose et al., 2004). In potato, the expression of nsLTP increases just

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prior to active metabolic processes, for example, tuberization, sprout development, dormancy breaking (Horvath et al., 2002), which are all processes involving source– sink relationships. Myzus persicae attack of cv. Kardal induced the expression of genes that are also induced in S. stoloniferum after M. euphorbiae attack (compatible interaction), but those genes are not induced in S. stoloniferum after the attack of M. persicae (incompatible interaction) (Alvarez et al., 2013). Among this group 3 transcripts are homologous to genes involved in molecule mobilization, that is, 2 hexose transporters, and 1 amino acid transport protein (AAT1). Two other transcripts are homologous to genes involved in protein catabolism; 1 is PR subtilisinserine-like protease P69, which is also activated in tomato plants upon Pseudomonas syringae infection, or treatment with SA or ET, (Jorda et al., 1999). The 2nd transcript is homologous to the chloroplast nucleoid DNA-binding protease 41 kD (CND41). This protease is involved in RuBisCO degradation and the translocation of nitrogen during senescence in tobacco (Kato et al., 2004). The CND41 is highly expressed in senescent leaves where chlorophyll loss is accompanied by the release and breakdown of RuBisCO (Wittenbach, 1979). Breakdown of the photosynthesis apparatus and subsequent mobilization of the breakdown products could result in nutritious enrichment of the phloem sap, which is advantageous to the aphid, as suggested by Zhu-Salzman et al. (2004). All the above mentioned genes could play a role in the acceptance of the plant as a host, since they are all genes related to turnover and mobilization of nutrients that can have beneficial effects on the aphid’s diet. Downregulated genes The transcription of most of the downregulated genes (31/38) was specific for old leaves of cv. Kardal attacked by M. persicae. Among them are homologues of genes related to secondary defense-compound biosynthesis, for example, two chalcone synthases, one chalconeflavonone isomerase, and one proteinase inhibitor (Table 1). In A. thaliana infested with M. persicae (compatible interaction) chalcone synthase genes are also downregulated (Moran et al., 2002), and in A. thaliana infested by B. brassicae chalcone synthase and chalcone–flavonone isomerase are downregulated as well (Kusnierczyk et al., 2008). Cultivar Kardal leaves, young and old, infested with M. persicae downregulate a transcript homologous to the dormancy/auxin repressed protein. We found the same in

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A. E. Alvarez et al.

S. stoloniferum after attack by M. euphorbiae or M. persicae (Alvarez et al., 2013). Although the physiological function of the gene is unknown, it is highly expressed in nongrowing buds of plants and downregulated in growing ones. Growing tissues have high sink strength (Herbers & Sonnewald, 1998), and the repression of this gene in leaves attacked by aphids could be related to the process of changing the physiological status of the tissue. In S. stoloniferum, aphid attack resulted in downregulation of a number of photosynthesis and photorespiration related genes (Alvarez et al., 2013). In cv. Kardal aphid attack did not trigger changes in expression of any of the photosynthesis and photorespiration related genes present on the microarray, neither in young nor in old leaf tissue. It is likely that in noninfested old leaves the expression level of these senescence-related genes is already reduced as a result of the developmental stage the leaves are in. Hence, the gene expression ratio for infested versus noninfested old leaves, which indicates relative transcript abundance, will be unchanged after aphid attack. We speculate that in cv. Kardal, the senescence of leaves contributes to the acceptance of the plant by the aphid. We do not know yet if M. persicae is able to induce senescence in mature leaves in cv. Kardal, but we noticed that leaves should be entering the senescence stage to be successfully infested by M. persicae. Perhaps the partial resistance present in cv. Kardal young leaves, similar to what Pierson et al. (2010) and Franzen et al. (2008) have found in other plant–aphid combinations, relies on the ability of the leaves to maintain the photosynthesis rate. Cottrell et al. (2009) found that black pecan aphid-induced leaf chlorosis plays an important role in the interaction of the aphid with its host. Aphid development can be retarded by applying plant growth regulators which retard chlorosis (Cottrell et al., 2010). Recently, Machado Assefh et al. (2013) showed that in S. tuberosum the induction of senescence contributes to the acceptance of the plant as a host for M. persicae. On young foliage of cv. Kardal aphid feeding is impeded as long as the leaves remains green. It is likely that young leaves have the capability to avoid infestation by retarding the changes that aphids induce at the feeding site, specially the changes related to senescence, but this has to be investigated further.

Acknowledgments This research was supported by the Alβan programme of the European Union (E03D16556AR) and by funding from the Ministry of Agriculture, Nature-management and Food quality of the Netherlands.  C 2013

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Accepted October 10, 2013

Institute of Zoology, Chinese Academy of Sciences, 21, 727–740