Pathways of amino acid degradation in Nilaparvata lugens (Stål) with special reference to lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH)

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Abstract

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.

Figures

  • Table 1. Counts of identified genes fromNi. lugens and YLS genome.
  • Table 1. (Continued)
  • Table 2. Primers used for RT-PCR, RACE, dsRNA synthesis and qRT-PCR.
  • Fig 1. Proposedmajor degradation pathways of amino acids inNilaparvata lugens. A) Pyruvate pathway; B) Oxaloacetate and fumarate pathway; C) α-Ketoglutarate pathway; D) Succinyl-CoAand acetyl-CoA pathway; and E) Acetoacetate pathway. The black arrows indicate reactions supported by genome and/or transcriptome annotation data. The numbers represent EC numbers. See Table 1 for details of the enzymes. Enzymes encoded by YLS genome are marked with white numbers in black boxes; the enzymes encoded by Ni. lugens genome are denoted with black number in grey boxes; the enzymes are homologous to those from bothNi. lugens and YLS genomes are given with black numbers without boxes; and the enzymes not found inNi. lugens and YLS genome and transcriptome data are marked with black numbers in dashed boxes.
  • Fig 2. The number of enzymes from YLS andNi. lugens (BPH) genome, respectively, involved in amino acid degradation. A) Pyruvate pathway; B) Oxaloacetate and fumarate pathway; C) α-Ketoglutarate pathway; D) Succinyl-CoAand acetyl-CoA pathway; and E) Acetoacetate pathway. Numbers in parentheses represent the number of enzymes from BPH or YLS, respectively. Numbers in the overlapping sections denote the number of enzymes originated from both BPH and YLS.
  • Fig 4. Phylogenic analysis of insect LKR/SDH. A rooted phylogenetic tree constructed by the maximum-likelihood method (the best-fitting model, LG+I+γ, with empirical frequency) based on the protein sequence alignments. The LKR/SDH-like sequences originated from Ni. lugens (N_lug), Rhodnius prolixus (R_pro, RPTMP07240), Acyrthosiphon pisum (A_pis LKR/SDH1, ACYPI004937; A_pis LKR/SDH2, ACYPI065217), Pediculus humanus (P_hum, PHUM016080), Tribolium castaneum (T_cas, TC002311), Nasonia vitripennis (N_vit, Nasvi2EG008769), Acromyrmex echinatior (A_ech, AECH18409), Atta cephalotes (A_cep, ACEP10511), Camponotus floridanus (C_flo, CFLO20285), Harpegnathos saltator (H_sal LKR/SDH1, HSAL12725; H_sal LKR/SDH2, HSAL12725), Bombus impatiens (B_imp, XP_003487153), Bombus terrestris (B_ter, XP_003398633), Apis mellifera (A_mel, GB47970), Apis florea (A_flo, XP_003692916), Aedes aegypti (A_aeg, AAEL014734), Anopheles gambiae (A_gam, AGAP008632), Culex quinquefasciatus (C_qui, CPIJ000416), Drosophila melanogaster (D_mel, FBgn0025687), Bombyx mori (B_mor, BGIBMGA010338),Heliconius melpomene (H_mel, HMEL016438), Plutella xylostella (P_xyl LKR/SDH1, Px002140; P_xyl LKR/SDH2, Px002187) and Danaus plexippus (D_ple, DPOGS208487).The LKR/SDH from Ixodes scapularis (I_sca, ISCW008489) was added as an outgroup. The percentiles of bootstrap values (1,000 replicates) are indicated. The scale bar represents the amino acid divergence. The pseudogenes are marked with asterisk.
  • Fig 5. Temporal (A) and spatial (B) expression patterns of the putativeNllkr/sdh. cDNA templates were derived from eggs, first-, second-, third- and fourth-instar nymphs (1st, 2nd, 3rd, 4th, 5th), and adults, or from ventral ganglion (VG), thorax muscles (TM), epidermis (EP), fat body (FB), gut (GU) and ovary (OV) of adults. For each sample, 3 independent pools of 5–10 individuals were measured in technical triplicate using qRT-PCR. The values are calculated using the 2-ΔΔCT method. The columns represent averages with vertical bars indicating SE.
  • Fig 6. Ingestion of dsNllkr/sdh on the mRNA level ofNllkr/sdh (A) and the survival (B) of the planthoppers. For the mRNA level, three biological replicates were conducted, and the mean ± SD (n = 3) was calculated to measure the relative transcript levels using the 2−ΔΔCT method. For both the mRNA level and accumulative mortality, the columns represent averages with vertical lines indicating SE. Columns that do not share the same letter are significantly different at P value of 0.05.

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Wan, P. J., Yuan, S. Y., Tang, Y. H., Li, K. L., Yang, L., Fu, Q., & Li, G. Q. (2015). Pathways of amino acid degradation in Nilaparvata lugens (Stål) with special reference to lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). PLoS ONE, 10(5). https://doi.org/10.1371/journal.pone.0127789

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