Silencing of V‐ATPase‐E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

Abstract

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V‐ATPase subunit E (V‐ATP‐E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V‐ATP‐E from D. citri had high homology with proteins from other insects. V‐ATP‐E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V‐ATP‐E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V‐ATP‐E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double‐stranded RNA of D. citri V‐ATP‐E was safe to non‐target parasitic wasps. These results suggest that V‐ATP‐E is an effective RNAi target that can be used in D. citri control to block CLas infection.

The transcriptomic response of Hyphantria cunea (Drury) to the infection of Serratia marcescens Bizio based on full-length SMRT transcriptome sequencing

“…V-ATPase is a class of important transporters that play an important role in plasma membrane proton transport in various cell types and are important for cellular processes ( Wagner et al., 2004 ; Beyenbach and Wieczorek, 2006 ; Forgac, 2007 ; Hinton et al., 2009 ). Studies on many insects, including D. citri , Sphenophorus levis Vaurie, and Locusta migratoria (Linnaeus), have found that the interference of V-ATPase gene will affect the growth and survival of insects ( Mohan et al., 2021 ; Guo et al., 2022 ; Liu et al., 2022 ). Studies have shown that RNAi knockdown of V-ATPase in Monochamus alternatus Hope inhibited the expression of immunity-related genes, including lysozyme , TAK1 , and pelle ( Li et al., 2022 ).…”

Comprehensive Assessment of Reference Gene Expression within the Whitefly Dialeurodes citri Using RT-qPCR

“…Primer pairs specific to D. citri were designed using Premier 5 software (v5.0) (refer to Table 1 ). The PCR reaction mixture (25 µL) included LA Taq DNA polymerase (Takara, Japan), and the PCR program followed the protocol described in Guo et al [ 18 , 19 , 20 ]. Electrophoresis was run in TAE buffer (Beyotime, Shanghai, China) at 120 V for 20 min.…”

“…There are hundreds of housekeeping genes, with the most frequently used ones being Actin, GAPDH, Tubulin, 18SrRNA and 28SrRNA [ 9 , 13 , 14 , 15 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. However, numerous studies have demonstrated that the so-called constant expression of any one of these housekeeping genes is only a “range” of constant expression in a certain type of cell or under the effect of an experimental factor, while in other types of cells or under experimental conditions, there are variations, which can sometimes be a dozen-fold, tens of folds or even hundreds of folds of differences [ 12 , 16 , 18 , 19 , 20 , 21 , 25 , 26 , 27 , 30 , 31 ]. In addition, there is no absolute generalization of the internal reference genes; if they are used directly without screening, they may lead to wrong or even opposite conclusions concerning the target gene [ 12 , 14 ].…”

“…Real-time fluorescence quantitative PCR, a powerful method for detecting gene expression profiles [ 8 , 33 , 34 ], is not only used for determining gene expression patterns but is also widely used to analyze differences in gene expression under different conditions, such as different time periods or samples that have undergone different treatments. This technique is also commonly used for verifying the accuracy and reliability of high-throughput sequencing (RNA-Seq) data of the transcriptome [ 11 , 17 , 18 , 19 , 34 , 35 ].…”

“…SRP065471) of D. citri , including Actin (β-Actin), RPL13 (ribosomal protein L13), α-tubulin , 18S (18S ribosomal RNA), V-ATP-A , RPS18 (ribosomal protein S), EF1A (elongation factor 1 α ) and ArgK (arginine kinase) [ 17 , 18 , 19 , 20 , 27 , 28 , 29 , 30 , 31 , 32 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. V-ATP-A and α- tubulin have proved to be the most optimal reference genes for different developmental stages and tissues in studies on Coleomegilla maculata , and studies on Diaphorina citri have demonstrated that V-ATP-A is expressed very stably at all developmental stages [ 17 , 19 ]; in a Henosepilachna vigintioctopunctata study, RPS18 was regarded as the best reference gene for different temperatures [ 22 ]; and ArgK showed the highest applicability as a reference gene in Lysiphlebia japonica and Spodoptera litura [ 18 , 27 , 32 , 36 ]. In the Sesamia inferens endogenous gene screen, 18S was shown to be optimal for different developmental stages and for sex [ 37 ].…”