Molecular cloning and expression of three polygalacturonase cDNAs from the tarnished plant bug, Lygus lineolaris

Abstract

Three unique cDNAs encoding putative polygalacturonase enzymes were isolated from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The three nucleotide sequences were dissimilar to one another, but the deduced amino acid sequences were similar to each other and to other polygalacturonases from insects, fungi, plants, and bacteria. Four conserved segments characteristic of polygalacturonases were present, but with some notable semiconservative substitutions. Two of four expected disulfide bridge-forming cysteine pairs were present. All three inferred protein translations included predicted signal sequences of 17 to 20 amino acids. Amplification of genomic DNA identified an intron in one of the genes, Llpg1, in the 5' untranslated region. Semiquantitative RT-PCR revealed expression in all stages of the insect except the eggs. Expression in adults, male and female, was highly variable, indicating a family of highly inducible and diverse enzymes adapted to the generalist polyphagous nature of this important pest.

Figure 1.

Genomic sequence of Lygus lineolaris polygalacturonase1 (PG1). Lowercase letters denote intron in the 5′ untranslated region. Start and stop codons are indicated in bold. Putative polyadenylation signal is underscored. GenBank accession number EU136628.

Figure 2.

Multiple sequence alignments of the deduced amino acid sequences. (A) L. lineolaris polygalacturonase (PG) cDNAs. ^* marks identities, : marks functionally conservative substitutions, . marks majority (2 of 3 either identical or functionally conserved). Predicted signal peptides are underscored. Conserved functional motifs are boxed, cysteines are shaded and lines connect putative disulfide bridges. Strictly conserved Tyr281 is shaded. (B) Multiple alignment of fungal and insect PGs.

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Figure 3.

Phylogenetic tree of some polygalacturonase proteins constructed by neighbor-joining (NJ) criteria. Bootstrap support values are shown at nodes, scale is 0.1 amino acid substitution per site. GenBank accession numbers and full species names for each sequence can be found in Table 2.

Figure 4

Expression profiles of polygalacturonase genes of Lygus lineolaris. (A-C) Relative semiquantitative RT-PCR values, as determined by gel density analysis and normalized to cytoplasmic actin, 16S ribosomal, and S2 ribosomal standards. Vertical bars connect the two expression levels in separate experimental replicates, horizontal crossbars indicate the average. EggD3: 3 day-old eggs, eggD8: 8 day-old eggs, nymphI2: second instar (small) nymphs, nymphMI5: fifth instar male nymph, nymphFI5: fifth instar female nymph, adultM: male adult, adultF: female adult. (A) Llpg1, (B) Llpg2, (C) Llpg3. (D) Semiquantitative RT-PCR gels of additional adult specimens. F: female, M: male, O: no template, PG1: Llpg1, PG2: Llpg2, PGs: Llpg3, S2: S2 ribosomal standard.

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