Annotation of chitin biosynthesis genes in Diaphorina citri, the Asian citrus psyllid

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.

. Annotation protocol for psyllid genome curation [16]. https://www.protocols.io/widgets/doi?uri=dx.doi. org/10.17504/protocols.io.bniimcce to heterogeneity of the sequenced psyllids, the genome has numerous false duplications of varying sizes, ranging from multiple adjacent genes to partial exons. As with all genomes, computationally annotated models provide a starting point, but often require manual correction.
We identified and manually annotated one CHS gene and two UAP genes in the D. citri genome v3. Although most insects have two CHS genes [3, 4] (Table 1), the presence of a single CHS gene is consistent with reports from other hemipteran genomes [5]. In contrast, D. citri seems to be unusual in that it has two UAP genes. Available RNA-seq data indicate that one of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our manual annotation of these chitin biosynthesis genes provides more accurate information for the design of future experiments involving these genes.  Multiple alignments of the predicted D. citri proteins and their insect homologs were performed using MUSCLE (RRID:SCR_011812) [17] or CLUSTALW (RRID:SCR_002909) [18] within MEGAX (MEGA software, RRID:SCR_000667), as specified in each figure legend.

METHODS
Phylogenetic trees were constructed using full-length protein sequences in MEGAX.

DATA VALIDATION AND QUALITY CONTROL Chitin synthases
Chitin synthases are the only enzymes in the chitin biosynthetic pathway that act specifically in the synthesis of chitin. This makes them an attractive, insect-specific target for RNA interference (RNAi)-based insecticides. The two CHS genes found in most Gigabyte, 2021, DOI: 10.46471/gigabyte.23  RNAi knockdown of CHS in D. citri causes increased lethality [30,33], supporting the idea that this gene is a good target for pest control.

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Our searches of the D. citri v3 genome revealed the previously described CHS gene, but no additional chitin synthase orthologs (Table 1). Transcriptomic evidence supports the existence of two CHS isoforms ( Table 4)   MCOT: MAKER (MAKER, RRID:SCR_005309), Cufflinks (Cufflinks, RRID:SCR_014597), Oases (Oases, RRID:SCR_011896), Trinity (Trinity, RRID:SCR_013048) pipeline. Each manually annotated gene has been assigned an OGSv3 gene identifier and denoted as a partial or complete model based on available evidence. Evidence types used for manual annotation are shown for each gene. A description of the various evidence sources and their strengths and weaknesses is included in our online protocol [16].
Greening Expression Network (CGEN), which contains RNA-seq data sets for various life stages and tissues [19]. Data from whole body samples indicate that CHS1 is expressed at all life stages, but is most highly expressed in juvenile stages (Figure 4). Our manual annotation of CHS1 corrects several errors that were present in the previous computationally predicted annotation for D. citri CHS (XP_017303059). Changes to the model include the addition of formerly missing sequence and the removal of artifactually duplicated regions. Domain analysis with TMHMM Server (TMHMM Server, RRID:SCR_014935, v2.0) indicates that the corrected CHS1-RA and CHS1-RB proteins have 15 transmembrane helices, as is typical for insect CHS proteins, rather than the 14 that were reported for the earlier version of the protein [30].

UDP-N-acetylglucosamine pyrophosphorylase (UAP)
In addition to its role in chitin synthesis, UAP is involved in the modification of other carbohydrates, sphingolipids and proteins. In Drosophila, mutants of UAP (also called  [17]. Individual amino acid alignments are denoted as identical (*), highly similar (:) or similar (.). Residues important for substrate binding by human UAP1 and conserved in T. castaneum are shaded according to their level of conservation. Identical residues are shaded blue and non-identical (but similar) residues are shaded red. The green shaded residue denotes the position of an alanine important for substrate binding in human UAP1 that is a cysteine in T. castaneum and other insects. named the D. citri genes UAP1 and UAP2, but no implication is intended of direct orthology with duplicated UAP genes in other insects.
We compared available expression data from the two D. citri UAP genes using CGEN [19].  [19]. Tissue types are shown on the X axis and expression levels (TPM) on the Y-axis. Blue bars denote expression levels in males and orange bars denote expression levels in females (all single replicate data). RNA-seq data from tissues labeled Wu et al. were sequenced in [22]. Data for the remaining tissues are from NCBI BioProject PRJNA448935. currently available should be interpreted with caution. Experimental analysis is outside the scope of this data release, but additional studies of UAP1 and UAP2 expression and function in individual males and females will be necessary to verify these results.

RE-USE POTENTIAL
There is considerable interest in use of the genes described here as targets for pest control.
At least two groups have already begun functional studies of the CHS gene in D. citri. Our improved annotations will allow more detailed experiments to be performed in the future.
For example, isoform-specific RNAi experiments on the CHS gene could be designed to determine the function of each transcript variant. The revised gene models will be incorporated into a new official gene set, which will be available for BLAST analysis and expression profiling on the Citrus Greening website [42] and the CGEN [19].

DATA AVAILABILITY
The Diaphorina citri genome assembly, official gene sets, and transcriptome data are

EDITOR'S NOTE
This article is one of a series of Data Releases crediting the outputs of a student-focused and community-driven manual annotation project curating gene models and if required, correcting assembly anomalies, for the Diaphorina citri genome project [2].