Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri

Abstract

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Keywords: CTV; Cathepsin B; Cathepsin L; Diaphorina citri; Huanglongbing; RNAi.

Figure 1

Expression of Cathepsin B in Diaphorina citri adults. The temporal pattern of Cathepsin B transcript relative expression in D. citri females (A) and males (B) was normalized to the expression value of the β-actin transcript. Data represent means ± standard deviations (SD) of 4 independent replicates. Asterisks indicate statistical differences between gene expression in psyllids fed on dsRNA-DCcathB or control plants using one-way ANOVA with Tukey’s post hoc test. N.S denotes no significance.

Figure 2

Expression of Cathepsin L in Diaphorina citri adults. The temporal pattern of Cathepsin L transcript relative expression in D. citri females (A) and males (B) was normalized to the expression value of the β-actin transcript. Data represent means ± standard deviations (SD) of 4 independent replicates. Asterisks indicate statistical differences between gene expression in psyllids fed on dsRNA- DcCathL or control plants using one-way ANOVA with Tukey’s post hoc test. N.S denotes no significance.

Figure 3

Effects of CTV-based RNAi (DCcathB and DCcathL) via expression in citrus plants on D. citri female reproductive development. (A) Temporal pattern of Vg1-like transcript relative expression levels in D. citri females normalized to the expression value of the β-actin transcript. Data represent means ± standard deviations (SD) of 4 independent replicates. Asterisks indicate statistical differences between gene expression in psyllids fed on plants expressing CTV-based dsRNAs (DcCathB and DcCathL) or control plants using one-way ANOVA with Tukey’s post hoc test. N.S denotes no significance. (B) Number of developing oocytes detected in D citri females after 10 days of challenge to plants expressing CTV-based dsRNAs or control plants. Data represents means ± SD (standard deviation) of at least thirty dissected females per group. (C) Representative images of ovaries from D. citri females. Scale bar is equal to 100 µm.

Figure 4

Hypothesis of Vitellogenin (Vg) and Cathepsins B and L-like (DcCathB and DcCathL) relationship. Vitellogenin (Vg) downregulated regarding DcCathB and DcCathL gene silencing resulting in decreased number of developing oocytes as well an ovarian development compromised.