Article Figures & Data
Figures
- Fig. 1.
Mean (±sd) expression ratios from microarrays with eight replicate cDNA spots of partial sequences ofN. attenuata Thr deaminase (TD; note break in yaxis and that only + sd are shown), proteinase inhibitor (PI), PMT, and RuBPCase small subunit genes hybridized with fluorescently labeled probes derived from M. sexta-attacked or control N. attenuata plants (harvested 2, 6, 12, or 24 h after the start of attack). Shaded area represents arbitrarily defined zone of nonsignificant changes in expression.
- Fig. 2.
Mean (±sd) expression ratios from microarrays with eight replicate cDNA spots with partial sequences of genes mediating N. attenuata's oxylipin cascade (lipoxygenase [LOX], HPL, AOS, and α-DIOX) hybridized with fluorescently labeled probes derived from M. sexta-attacked or control N. attenuata plants (harvested 2, 6, 12, or 24 h after the start of attack). Shaded area represents arbitrarily defined zone of nonsignificant changes in expression.
- Fig. 3.
Mean (±sd) expression ratios from microarrays with eight replicate cDNA spots with partial sequences ofN. attenuata xyloglucan (XG) endo-transglycosylase (XTH1), basic pathogenesis-related protein (PR1), HMGR, and EAS genes hybridized with fluorescently labeled probes derived from M. sexta-attacked or control N. attenuata plants (harvested 2, 6, 12, or 24 h after the start of attack). Shaded area represents arbitrarily defined zone of nonsignificant changes in expression.
- Fig. 4.
Alignment of deduced amino acid sequences ofN. attenuata HMGR (AF542543), N. sylvestris(S24760), N. tabacum (AAB87727), and pepper (Q9XEL8). Missing amino acids are indicated by dashes, and different amino acids are indicated by black shading. A transmembrane domain with two segments located in the N-terminal region and a 349-amino acid residue catalytic domain containing three highly conserved signatures, located in the center of the catalytic domain (1), in a Gly-rich region (2), and in a region containing a His residue (3), are indicated by boxes and gray shading.
- Fig. 5.
Alignment of deduced amino acid sequences of EAS from different genotypes of N. attenuata (EASutah[AF542544], EAS12 [AF484123], EAS34[AF484124], and EAS37 [AF484124]) and N. tabacum (5EAS). Amino acid differences are indicated by black shading. The gene consists of two domains: domain 1 (enclosed in box with a dotted line), which bisects domain 2 into two parts (both enclosed in boxes with a solid lines). Two conserved Mg2+-binding sites located in C-terminal portion of domain 2 are indicated by gray shading.
Tables
- Table I.
Genes cloned by DDRT-PCR (DD/arbitrary primer no.) and subtractive hybridization with magnetic beads (SHMB) that exhibited nonsignificant (between 0.5 and 1.50) expression ratios in the microarray analysis and had similarity to genes in the databases
Clone Length Method Peak at h Expression Ratio Type Accession No. Sequence Similarity E Value bp DH017 1028 SHMB 24 1.33 IIb CA591807 Matrix attachment regions-binding protein (MARBPF;AB059832) 0.00 DH108 453 SHMB 12 0.80 III CA591793 PSII (NtPII10; X70088) 0.00 RE322 590 DD/R5 24 1.25 Ia CA591771 Transcription factor NtWRKY2 (AB020590) 1 × 10–120 DH123 371 SHMB 24 1.21 IIa CA591796 Cytosolic glyceraldehyde-3-phosphophate dehydrogenase (GapC; M14419) 2 × 10–94 RE283 184 DD/R5 12 0.63 III CA591769 PSII oxygen-evolving complex, 23-kD polypeptide (X58910) 1 × 10–73 RD161 234 DD/R4 12 1.47 III CA591756 Actin gene Sfa 15B (X03076) 1 × 10–47 RB142 107 DD/R2 24 1.13 IIb CA591717 25S ribosomal RNA gene (X13557) 1 × 10–43 RF064 161 DD/R6 24 1.20 IIb CA591783 26S ribosomal RNA gene (AF479172) 2 × 10–43 RD131 190 DD/R4 12 1.30 III CA591754 Signal recognition particle 7S RNA (Z29099) 3 × 10–35 DH270 307 SHMB 6, 24 1.20, 1.23 IV CA591818 Type 2 metallothionein-like protein (U35225) 6 × 10–31 RN021 389 DD/R14 24 1.43 Ia CA591773 Beta-alanine synthase (Y19104) 3 × 10–30 RN032 337 DD/R14 12 0.54 III CA591774 Polypeptide of PSII (X85038) 1 × 10–17 RC231 276 DD/R3 12 1.23 III CA591748 myb1 gene (AF248962) 8 × 10–17 Genes are listed in order of decreasing E value from BLAST queries. Expression patterns are defined as: gradual (Type Ia) or abrupt (Type Ib) increases; initial decreases followed by either steady (Type IIa) or abrupt increases (Type IIb); an initial increase followed by a decrease (Type III); an increase, a decrease, and finally an increase (Type IV; both peaks are given if equal) as plants were continuously attacked by Manduca sexta larvae over 24 h. Down-regulated genes have the opposite patterns.
- Table II.
Genes cloned by DDRT-PCR (DD/arbitrary primer no.) and subtractive hybridization with magnetic beads (SHMB) that exhibited significant (≤0.5 and ≥1.50) expression ratios in the microarray analysis and had similarity to genes in the databases
Clone Length Method Peak at h Expression Ratio Type Accession No. Sequence Similarity E Value bp DH120 394 SHMB 24 2.38 IIa AF542543 3-Hydroxy-3-methylglutaryl-CoA reductase (X63649) 0 DH164 452 SHMB 24 1.58 IIa AF542544 5-Epi-aristolochene synthase mRNA (AF272244) 1 × 10–168 DH114 372 SHMB 2 0.40 Ia CA591794 26S Ribosomal RNA gene spacer (Y08427) 1 × 10–156 DH083 607 SHMB 6, 24 1.66, 168 IV CA591811 Tumor-related protein (D26464) 1 × 10–137 RN254 487 DD/R14 24 1.90 Ia CA591779 Ubiquitin carrier protein (Ubc-E2) mRNA (L23762) 1 × 10–120 DH099 482 SHMB 24 3.34 Ia CA591812 Basic pathogenesis-related protein (PR1; X14065) 1 × 10–115 RN161 248 DD/R14 24 2.11 Ia CA591777 Transformer-2-like SR ribonucleoprotein (RNP; Y09506) 1 × 10–113 DH104 594 SHMB 24 1.66 IIa CA591822 Thiazole biosynthetic enzyme precursor (NM 124858) 1 × 10–86 DH054 224 SHMB 24 2.04 Ia CA591790 Sulfite reductase (AB010717) 2 × 10–73 RB271 484 DD/R2 12 2.33 IV CA591719 Xyloglucan endo-transglycosylase (X82685) 2 × 10–77 RB521 484 DD/R2 24 3.44 IV CA591731 Xyloglucan endo-transglycosylase (X82685) 1 × 10–75 RB061 230 DD/R2 24 2.88 Ia CA591715 13-Lipoxygenase clone H3 (X96406) 2 × 10–72 RC191 233 DD/R3 24 2.70 Ia CA591745 ant Gene for ATP/ADP translocator (X62123) 8 × 10–72 RB131 427 DD/R2 24 1.51 IIb CA591716 Allene oxide synthase (AJ457080) 3 × 10–65 DH162 461 SHMB 24 2.21 Ia CA591801 RNA-binding Gly-rich protein (RGP-1a; D16204) 1 × 10–61 RF113 561 DD/R6 24 2.31 IIa CA591788 Cytokinin-induced (cig2) mRNA (AB031321) 9 × 10–57 RB493 231 DD/R2 2 1.91 IIa CA591728 (Zymonaonas mobilis) rrnB operon (AF088897) 1 × 10–55 DH283 353 SHMB 12 1.64 III CA591810 Chaperonin 60 (X70868) 2 × 10–55 DH124 586 SHMB 24 1.63 IIa CA591814 (Nicotiana tabacum) GTP-binding protein (Ran-A1) mRNA (L16767) 5 × 10–44 RF071 255 DD/R6 12 0.50 IIa CA591784 Ribulose-1,5-biphosphate carboxylase small subunit (M32420) 1 × 10–33 RB012 289 DD/R2 24 1.51 IIa CA591712 Alpha-amylase (amy21) mRNA (M81682) 1 × 10–27 DH219 341 SHMB 24 1.76 Ia CA591804 Histone H3 (PcH3–20) gene (M77494) 1 × 10–18 DH193 359 SHMB 24 4.96 Ib CA591816 Major intrinsic protein (MIP) 2 (Y18312) 7 × 10–18 DH182 627 SHMB 24 3.65 Ib CA591802 Ser carboxypeptidase cp-b (AJ 251970) 2 × 10–16 RB481 535 DD/R2 24 1.79 Ia CA591726 (Arabidopsis) putative protein (MN_127327) 1 × 10–11 DH138 295 SHMB 24 2.09 IIa CA591799 60S ribosomal protein (NM_114851) 2 × 10–11 RB304 425 DD/R2 24 1.67 IIa CA591720 (Lotus japonicus) genomic DNA, chromosome 6 (AP004526) 2 × 10–09 RE234 326 DD/R5 2 1.69 IIa CA591767 Plastidic aldolase-like protein (AB027001) 3 × 10–08 RE112 345 DD/R5 24 4.38 Ia CA591761 Ser protease sbt4b (AJ006480) 6 × 10–06 RC144 342 DD/R3 12 1.83 IIb CA591742 VFNT Pto locus (AF220603) 7 × 10–06 DH061 399 SHMB 24 1.70 Ia CA591791 (Arabidopsis) putative protein, mRNA (MN_117913) 9 × 10–06 DH126 302 SHMB 12 0.43 IIa CA591815 ATP-binding cassette (ABC) transporter protein 1 (MN_125882) 1 × 10–3 RC095 496 DD/R3 24 2.41 Ia CA591780 Ubiquitin carrier protein (Ubc) mRNA (L23762) 0.040 Genes are listed in order of decreasing E value from the BLAST queries. Expression patterns are defined as: gradual (Type Ia) or abrupt (Type Ib) increases; initial decreases followed by either steady (Type IIa) or abrupt increases (Type IIb); an initial increase followed by a decrease (Type III); an increase, a decrease, and finally an increase (Type I;: both peaks are given if equal) as plants were continuously attacked by M. sexta larvae over 24 h. Down-regulated genes have the opposite patterns.
