Supplementary results
Commonly regulated genes. Apparently, the classical pathways mediating
resistance to insects and pathogens are activated by both insect species; these include the
octadecanoid- (13-LOX, OPR3, JMT), the salicylic acid- (PAL, SMT), and the ethylene
pathway (ACO). Another induced gene encodes -dioxygenase, which produces 2hydroyperoxides that may act as precursors for oxylipin signals or other products toxic to
pathogens (Hamberg et al., 1999). Several direct defense genes are up-regulated, such as
serin- and trypsin inhibitors, which interfere with herbivores’ gut proteases (confirming
previous results with Northern analysis, Voelckel and Baldwin, 2003); SAMDC, which
converts putrescine to polyamines - in free or conjugated forms polyamines are involved
in plant defense (refs in Voelckel and Baldwin, 2003); PMT, which converts putrescine
to N-methylputrescine - this acts as a precursor for nicotine and tropan alkaloid synthesis;
and key genes in phenylpropanoid metabolism (PAL, C4H, 4CL), which lead to the
production of cinnamic- and coumaric acid – these acids constitute precursors for the
biosynthesis of flavanoids, lignins, hydroxycinnamic acid amides and other phenolics.
The activation of a rhamnosyltransferase, which catalyzes the formation of rutin, the
rutinoside of quercetin-3-glucoside, may act in concert with the stimulation of the
synthesis of the flavanoid quercetin. The following genes are also up-regulated:
polyphenoloxidase, involved in tomato defense against Pseudomonas syringae (Li and
Steffens, 2002); a pto-responsive gene, presumably involved in pto-mediated defenses
against pathogens (Accession nr.: AF146690); and glutathione peroxidase, an enzyme
that alleviates cellular oxidative stress and increases in response to fungal pathogens
(Herbette et. al, 2003); in contrast, the pathogenesis-related proteins 2, 4, and 5 are downregulated. More commonly up-regulated genes are hydroperoxide lyase, which catalyzes
the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids (e.g. C6-volatiles,
Vancanneyt et al., 2001); an SNF1-related kinase, which plays a role in nutritional and
environmental stress responses (Bradford et al., 2003); a mitogen-activated protein
(MAP) kinase; which is part of the wound response in tobacco (WIPK, Seo et al., 1995),
and xyloglucan-endotransglycosylases (XTH), which cleave and transfer xyloglucan
molecules and mediate loosening/fortification of cell walls. Commonly down-regulated
photosynthesis genes include genes of the electron transport pathway (69-72) and
generation of NADPH (80) as well as genes involved in CO2 assimilation (59-68, 73-76,
78, 81) and carbohydrate transport (79). Moreover, glycine hydroxymethyltransferase,
which is involved in serine biosynthesis and in recouping carbon in photorespiration;
glutamine synthetase (GS), which produces glutamine from glutamate and ammonium in
plastids; phospholipase 2, which catalyzes the formation of diacylglycerol, a second
messenger; an RNA-binding glycine rich protein, which is likely involved in processing
of pre-mRNA (Moriguchi et al., 1997); the biosynthetic enzyme for thiazole, the
precursor of thiamine, which acts a co-factor for some citric acid cycle enzymes; a
germin-like protein, which may play a role in pathogen defense (Membre et al., 2000);
and a transcript with similarity to the H3 class of histone proteins, are non-specifically
down-regulated. Nine unknown genes are up- and seven unknown genes are downregulated in response to both herbivores.
Specifically regulated genes. According to our criteria, 117 oligo-probes revealed
differential expression of the respective genes between M. sexta- and T. notatus-attack.
However, only a few genes seem to be truly differentially expressed. Since many genes
are represented by more than one probe (ExpII-SupplMat1), genes for which some probes
indicate specific expression (e.g. ACO up-regulation by M. sexta) but others indicate
nonspecific expression (e.g. ACO-up-regulation by M. sexta and T. notatus) are not
assumed to be differentially expressed. These would include SNF1, ACO, XTH, PPO, adioxygenase, 13-LOX, WIPK, PR 4, GS, glyceraldehyde-3-phophate-dehydrogenase, and
phosphoglycerate kinase. Moreover, genes for which one probe indicates M. sextaspecific but another indicates T. notatus–specific expression (e.g. up-regulation of AOS,
a gene from the jasmonate cascade) are considered to be commonly expressed. Similarly,
threonine deaminase (TD), which catalyzes the committed step in isoleucine biosynthesis
(Samach et al., 1991) and which seems to be up-regulated only by T. notatus-attack (does
not fulfill t-test criterion for M. sexta), is not considered to be mirid-specific. On the
contrary, TD expression was clearly enhanced by both herbivores on the cDNA array
(62fold increase after M. sexta.-, 18fold after T. notatus-attack) and in previous studies
was found to be M. sexta-induced (Hermsmeier et al., 2001; Schittko et al., 2001).
Another gene considered commonly induced codes for thionin. Thionins are
antimicrobial compounds, which may exhibit amylase- or proteinase-inhibitor activity
and have been shown to be both M. sexta- and mirid-induced in prior Northern analysis
(Voelckel and Baldwin, 2003 and refs therein). Here their mirid-induced ERs marginally
miss the 1.5 arbitrary threshold but differ significantly from 1…
… More genes specifically up-regulated by M. sexta are a calcium-dependent
protein kinase and a wound-stimulated protein, both of which have been implicated in
plant defense against pathogens (Romeis et al., 2001; Pozueta-Romero et al., 1995).
More genes specifically down-regulated by M. sexta include an enzyme providing
reduced thioredoxin (55), which by reducing disulfide bonds of other primary metabolism
enzymes modulates their activity; a chloroplastic outer envelope protein selective for
amino acids (Pohlmeyer et al., 1997); two genes from the protein translation machinery,
β-farnesene synthase, which converts farnesylpyrophosphate (FPP) to acyclic
sesquiterpenes (Deligeorgopoulou and Alleman, 2003); mitochondrial chaperonin 60, and
a transformer-2-like ribonucleoprotein, which functions in constitutive and alternative
splicing of nuclear pre-mRNA.
Genes with inconsistent responses. While all six N. attenuata-derived probes for
the small subunit of rubisco (ssu) indicate its down-regulation after herbivore attack, a L.
esculentum-derived ssu probe indicates its up-regulation. The sequences that best match
the Nicotiana oligos (ssu from N. sylvstris and ssu pseudogene from N. tabacum) show
almost no similarity to the ssu oligo template sequence from L. esculentum, indicating
large differences between the ssu gene family in tobacco and tomato. While all upregulated XTH gene probes are similar to the largely identical XTH1 and XTH2 genes
from tomato, the XTH probe down-regulated by M. sexta was derived from tomato
XTH4, indicating different induction of XTH isoforms. All sucrose-phosphate synthase
(SPS) probes were designed after two largely identical sequences from potato and tomato,
but show up- as well as down-regulation in response to insect attack. Therefore, parts of
the genes must be different in N. attenuata. Similarly inconsistent results are obtained
with probes for luminal binding protein (BiP) genes, which are activated during ER stress
(Denecke et al., 1991), and a 5-epi-aristolochene synthase (EAS) gene, which is an FPP
cyclase involved in the formation of the antimicrobial compound capsidiol (Bohlmann et
al., 2002). In both cases probes derived from N. attenuata sequences yielded a regulation
pattern different from probes from N. tabacum sequences indicating different gene
structures between the two tobacco species. Unless the gene structures for SPS, BiP, and
EAS have been elucidated in N. attenuata, their regulation after herbivore attack can not
be reliably inferred from these data.
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