Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

Abstract

Background: Infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data.

Results: In total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters.

Conclusions: Altogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.

Keywords: Enzymatic activity; Gut; Proteomics; RNA-Seq; Salivary gland; Tea green leafhopper.

Fig. 1

Enzymatic activity of selected proteases in homogenates of salivary glands and guts of E. onukii. Error bars indicate the standard deviation from the mean for three replications. Statistical comparisons were conducted between the enzymatic activity of salivary glands and guts (*0.01 < P < 0.05, **P < 0.01, Student’s t-test)

Fig. 2

Summary of proteins mapped to proteomic peptides. Panel a Number of proteins identified in the SG, gut or both tissues. Panel b Correlations between transcript abundance and the probability of proteins mapped to proteomic peptides. Translated protein sequence sets derived from the salivary glands and gut were respectively used as targets for mapping of peptides that resulted from the proteomic analysis of the corresponding tissue. The proportion of proteins detected from the proteome increased with increasing FPKM values

Fig. 3

Transcriptomic abundance of proteases mapped to proteomic peptides. Panel a Median FPKM of predicted proteins in the SG and gut of E. onukii. Error bars indicate the standard deviation of the median FPKM of proteins in three biological replications. Panel b Number of proteases identified in the SG, gut or both tissues

Fig. 4

Phylogenetic tree containing aminopeptidases from six hemipteran species. Protein sequences of aminopeptidase-like proteins from Nilaparvata lugens (Nl), Laodelphax striatellus (Ls), Nephotettix cincticeps (Nc), Acyrthosiphum pisim (Ap), Halyomorpha halys (Hh) and Empoasca (Matsumurasca) onukii (EMo) were aligned and used for the phylogenetic analysis through the maximum likelihood strategy (Panel a). Information regarding the proteins is shown in Files S3 and S6. Proteins from E. onukii are indicated in bold. Proteins in the phylogenetic tree mentioned in the paper are arrowed. Groups A, B, F and G: proteins annotated by aminopeptidase N; Group C: glutamyl aminopeptidase; Group D: puromycin sensitive aminopeptidase; Group E: endoplasmic reticulum aminopeptidase; Group H: methionine aminopeptidase; Group I: xaa-Pro aminopeptidase; Group J: aminopeptidase NPEPL1; Group K: cytosol aminopeptidase; Group L: aminopeptidase W07G4.4e. Relative expression level is presented by log₂FC (gut vs SG) and shown by a colored circle outside the tree, from green (log₂FC = −6) to red (log₂FC = 9). Proteins with no log₂FC of FPKM are left blank. The sequence logos (logs?) of the gluzincin motif and zinc-binding motif from aminopeptidase N-like proteins in groups A, B, F and G are shown in (panel b)

Fig. 5

Phylogenetic tree containing cathepsin B- and cathepsin L-like proteins from six hemipteran species. Protein sequences of cathepsin B- and cathepsin L-like proteins from N. lugens (Nl), L. striatellus (Ls), N. cincticeps (Nc), A. pisim (Ap), H. halys (Hh) and E. onukii (EMo) were aligned and used for the phylogenetic analysis through the maximum likelihood strategy. Cathepsin D-like proteins of N. lugens and L. striatellus were clustered as the outgroup. Information regarding the proteins is shown in Files S3 and S6. Proteins from E. onukii are indicated in bold. Proteins in the phylogenetic tree mentioned in the paper are arrowed. The clade highlighted by pink and yellow contains cathepsin B- and cathepsin L-like proteins, respectively. Relative expression level is presented by log₂FC (gut vs SG) and shown by a colored circle outside the tree, from green (log₂FC = −3) to red (log₂FC = 11). Proteins with no log₂FC of FPKM are left blank

Fig. 6

Phylogenetic tree containing serine protease-like proteins from six hemipteran species. Protein sequences annotated by trypsin, chymotrypsin and elastase from N. lugens (Nl), L. striatellus (Ls), N. cincticeps (Nc), A. pisim (Ap), H. halys (Hh) and E. onukii (EMo) were aligned and used for the phylogenetic analysis through the maximum likelihood strategy. Information regarding the proteins is shown in Files S3 and S6. Proteins from E. onukii are indicated in bold. Proteins in the phylogenetic tree mentioned in the paper are arrowed. Clades highlighted by blue and orange indicate two clusters of serine protease. The branch highlighted by red indicates a cluster of serine protease showing relatively higher FPKMs in the salivary gland than in the gut. Relative expression level is presented by log₂FC (gut vs SG) and shown by a colored circle outside the tree, from green (log₂FC = − 10) to red (log₂FC = 13). Proteins with no log₂FC of FPKM are left blank

Fig. 7

Excised salivary glands of Empoasca (Matsumurasca) onukii nymphs. Salivary glands were dissected and imaged by digital microscope VHX-2000C (KEYENCE, Japan). a-d indicate four lobes of one side of the salivary gland