Utilizing a chromosomal-length genome assembly to annotate the Wnt signaling pathway in the Asian citrus psyllid, Diaphorina citri

Abstract

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB), or citrus greening disease. This disease has devastated Florida's citrus industry, and threatens California's industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirms 24 Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with a gene configuration like that in Drosophila melanogaster. These findings provide greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can be used to develop molecular systems, such as CRISPR and RNAi, which target and control psyllid populations to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project.

Figure 1.

Theoretical model of canonical Wnt signaling cascade in D. citri based on curated genes. (1) Wnt is secreted. (2) Wnt concentration gradient forms. (3) Wnt binds to Frizzled and releases Armadillo. (4) Armadillo migrates into the nucleus, associates with transcription factor Pangolin, and regulates gene expression. (5) Armadillo is degraded in the absence of Wnt.

Figure 2.

Protocol for psyllid genome curation [17]. https://www.protocols.io/widgets/doi?uri=dx.doi.org/10.17504/protocols.io.bniimcce

Figure 3.

Neighbor-joining tree of Wnt protein sequences. Phylogenetic analysis was performed to categorize the seven D. citri Wnt genes (signified by dots). Wnt families are distinguished by clades and are color coded. Bootstrap values are based on 1000 replicates and values under 25 are removed. Ortholog sequences were collected from NCBI protein database [18]. Analysis was performed using MEGA7 [23].

Figure 4.

Wnt genes in six insects. A colored box indicates the presence of a Wnt subfamily (1–11, 16, and A) in that insect, while a white box indicates the loss of a subfamily. For example, all six species have Wnt1 and Wnt5, none have Wnt2-4, and only A. pisum has Wnt16. Homologs of Wnt8 in T. castaneum and D. melanogaster are also referred to as WntD.

Figure 5.

Wnt1-6-10 cluster comparison. Organization of the Wnt1-6-10 cluster in D. citri is similar to that of D. melanogaster and differs from what may be a basal arthropod gene arrangement seen in A. gambiae, T. castaneum, A. mellifera, and D. pulex. Gene lengths are not to scale.

Figure 6.

Transcript levels of clustered Wnt transcripts during different D. citri life stages. Whole body transcript expression data from egg, nymph, and adult stages [47] were collected from CGEN [48]. The psyllids were raised on Citrus macrophylla and were not infected with Candidatus Liberibacter asiaticus. Expression values shown in transcripts per million (TPM).

Figure 7.

Transcript levels of D. citri Wnt repertoire in both nymph and adult psyllids from whole body RNA extractions. Green bars indicate the average transcript levels for Wnt in nymph samples [47], and gray bars represent the average transcript levels for Wnt in adult samples. Averages are based on six nymph samples and six adult samples. Expression levels shown in transcripts per million (TPM). Standard deviation of samples is shown by error bars. RNA-seq data was collected from CGEN [48].

Figure 8.

Neighbor-joining tree of insect Frizzled protein sequences. Proteins grouped in the Frizzled 1 subfamily are highlighted in green, Frizzled 2 in orange, Frizzled 3 in blue, and Frizzled 4 in magenta. Circles indicate the D. citri sequences. Ortholog sequences were collected from the NCBI protein database [18]. Some NCBI sequences (such as XP_006568530.1, XP_008188372.2, and XP_022194032.1) may have numeric labels derived from computational predictions that do not reflect sequence or functional similarity. Analysis was performed using MEGA7 [23].

Figure 9.

Domain comparison of ROR1 isoforms. The immunoglobulin domain (IG_like) is present in isoform 1. Other shared domains include a cysteine-rich frizzled domain (CRD_FZ), a Kringle domain (KR), and a protein kinase catalytic domain (PKc_like). Domains were calculated and visualized using the NCBI Conserved Domain Architecture Retrieval Tool (CDART).

Figure 10.

Expression of ROR1 isoforms in egg, nymph and adult D. citri. Pink bars indicate the average transcript levels for isoform 1 (Dcitr05g14430.1.1), and orange bars indicate the average transcript levels for isoform 2 (Dcitr05g14430.1.2). Note: only one egg sample was used for comparison. Egg transcripts from the whole egg (one sample), nymph transcripts from the whole body (six samples), and adult transcripts from the whole body, abdomen, and thorax (14 samples) are shown. Expression values shown in transcripts per million (TPM). Data labels note the average TPM. Standard deviation of samples, when available, is shown by error bars. RNA-seq data was collected from CGEN [48].