viruses Article Cytochrome P450 Monooxygenases CYP6AY3 and CYP6CW1 Regulate Rice Black-Streaked Dwarf Virus Replication in Laodelphax striatellus (Fallén) Jian-Hua Zhang 1,2 , Ming Zhao 2, Yi-Jun Zhou 1, Qiu-Fang Xu 1,3,* and Yuan-Xue Yang 2,* 1 Key Laboratory of Food Quality and Safety of Jiangsu Province, State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; [email protected] (J.-H.Z.); [email protected] (Y.-J.Z.) 2 Institute of Industrial Crops, Shandong Academy of Agricultural Sciences, Jinan 250100, China; [email protected] 3 Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China * Correspondence: [email protected] (Q.-F.X.); [email protected] (Y.-X.Y.) Abstract: The small brown planthopper, Laodelphax striatellus (Fallén), is an important agricultural pest that causes significant losses by sucking and transmitting multiple plant viruses, such as rice black-streaked dwarf virus (RBSDV). Insecticides are commonly used to control planthoppers and cause the induction or overexpression of cytochrome P450 monooxygenases (P450s) from the CYP3 and CYP4 clades after insecticide application. However, little is known about the roles of insecticides and P450s in the regulation of viral replication in insects. In this study, RBSDV-infected L. striatellus were injected with imidacloprid, deltamethrin, pymetrozine, and buprofezin, respectively. The insecticide treatments caused a significant decrease in RBSDV abundance in L. striatellus. Treatment Citation: Zhang, J.-H.; Zhao, M.; of piperonyl butoxide (PBO), an effective inhibitor of P450s, significantly increased the RBSDV Zhou, Y.-J.; Xu, Q.-F.; Yang, Y.-X. abundance in L. striatellus. Fourteen P450 candidate genes in the CYP3 clade and 21 in the CYP4 clade Cytochrome P450 Monooxygenases were systematically identified in L. striatellus, and their expression patterns were analyzed under CYP6AY3 and CYP6CW1 Regulate RBSDV infection, in different tissues, and at different developmental stages. Among the thirty-five Rice Black-Streaked Dwarf Virus P450 genes, the expression level of CYP6CW1 was the highest, while CYP6AY3 was the lowest Replication in Laodelphax striatellus after RBSDV infection. Knockdown of CYP6CW1 and CYP6AY3 significantly increased the virus (Fallén). Viruses 2021, 13, 1576. abundance and promoted virus replication in L. striatellus. Overall, our data reveal that CYP6CW1 https://doi.org/10.3390/v13081576 and CYP6AY3 play a critical role in the regulation of virus replication in L. striatellus. Academic Editor: Yau-Heiu Hsu Keywords: insecticides; Laodelphax striatellus; rice black-streaked dwarf virus; P450s; CYP6CW1; Received: 28 June 2021 CYP6AY3; replication Accepted: 5 August 2021 Published: 10 August 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Rice production is severely affected by planthoppers, such as Laodelphax striatellus, published maps and institutional affil- Sogatella furcifera, and Nilaparvata lugens. The rice planthoppers can cause losses by feeding. iations. Besides, they also transmit a variety of plant viruses, causing severe yield losses in rice- producing regions. For example, S. furcifera can transmit Southern rice black-streaked dwarf virus, while N. lugens can transmit rice grassy stunt virus and rice ragged stunt virus [1,2]. Laodelphax striatellus can transmit rice stripe virus, rice black-streaked dwarf Copyright: © 2021 by the authors. virus (RBSDV), maize rough dwarf virus, northern cereal mosaic virus, and barley yellow Licensee MDPI, Basel, Switzerland. striate mosaic virus [3–7]. The outbreak of these viral diseases caused severe yield losses in This article is an open access article rice-producing regions [4]. Insecticides are widely used to control planthoppers. However, distributed under the terms and little is known about the effect of insecticide on the virus transmitted by the planthoppers. conditions of the Creative Commons Cytochrome P450 monooxygenases (P450) are a group of heme-thiolate enzymes that Attribution (CC BY) license (https:// catalyze a variety of reactions related to metabolism and chemical toxicity [8]. The P450s creativecommons.org/licenses/by/ are divided into CYP2, CYP3, CYP4, and mitochondrial CYP clades. A series of P450s 4.0/). Viruses 2021, 13, 1576. https://doi.org/10.3390/v13081576 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1576 2 of 13 in CYP3 and CYP4 clades are induced or overexpressed in planthoppers and involved in the metabolism of insecticides, such as CYP4CE1, CYP6AY1, and CYP6CW1 [9–11]. Metabolic resistance mediated by the P450s has been considered as the dominant factor for insecticide resistance in the field populations of planthoppers [12–14]. A previous study showed that the genetic variants of CYP2E1 and CYP1A1 resulted in the susceptibility to chronic Hepatitis B virus infection [15], suggesting P450s in CYP3 and CYP4 clades might be involved in the regulation of virus infection. Rice black-streaked dwarf virus is one of the important viruses transmitted by L. striatellus. It is the causal agent of rice black-streaked dwarf and maize rough dwarf disease, which cause considerable yield losses in East Asia [16]. RBSDV belongs to the genus Fijivirus of the family Reoviridae. Its genome includes ten double-stranded RNA (dsRNA) segments (S1 to S10) and encodes thirteen proteins. Each segment of S5, S7, and S9 encodes two proteins. P5-1, P6, and P9-1 proteins, which are encoded by the segments of S5-1, S6, and S9-1, are the components of the viroplasm [17–19]. RBSDV P10 protein, encoded by S10 segment, is the outer capsid protein, which plays a key role in viral infection process [20,21]. Midgut of L. striatellus is a barrier for RBSDV infection [22]. Currently, there are no rice cultivars resistant to RBSDV. Using insecticide to control L. striatellus population is still one of the important strategies to control the viral disease. High-efficiency, low-toxicity, and low-residue insecticides, such as mesoionic insecticide, triazinone insecticide, pyrethroid insecticide, neonicotinoid insecticide, and insect growth regulator, are most widely used to control planthoppers in the fields [23]. Imidacloprid is a representative neonicotinoid insecticide and can disturb the nervous system of insects [10,11], while deltamethrin is a modulator of nerve sodium channels in insects [24]. Pymetrozine is a fast-acting and selective inhibitor of the feeding of sap-sucking insects [14]. Buprofezin is an insect growth regulator and can interfere with metabolism and inhibit chitin synthesis in insects [14]. Fourteen P450s were overexpressed in the deltamethrin-resistant L. striatellus strain [24]. Overexpression of CYP6CW1 was involved in the resistance to buprofezin and pymetrozine in L. striatellus populations [14]. Imidacloprid treatment significantly upregulates the P450 gene CYP4C71 in L. striatellus [25]. The function of P450s in metabolism of insecticides in insects is well studied. However, whether P450 genes play a role in regulation of virus replication in L. striatellus is largely unknown. In this study, the effect of insecticides on viruses was investigated using RBSDV and L. striatellus interaction system. Four representative insecticides, including imidacloprid, deltamethrin, pymetrozine, and buprofezin, were selected to study whether insecticides affect RBSDV infection in L. striatellus. We showed that the application of these four insecticides reduced the RBSDV abundance in L. striatellus. Furthermore, inhibition of the P450s activities by P450s inhibitor piperonyl butoxide (PBO) significantly decreased the RBSDV abundance in L. striatellus. Knockdown of P450s CYP6AY3 and CYP6CW1 negatively regulated the RBSDV abundance and replication in L. striatellus. These results demonstrated that insecticides and P450s can regulate virus infection in the insects. 2. Materials and Methods 2.1. Insects The virus-free (VF) populations of L. striatellus were collected from Haian (32.57◦ N, 120.45◦ E; Jiangsu, China) in 2004 and maintained in the laboratory. The L. striatellus was reared on rice seedlings at 25 ± 1 ◦C with 70−80% humidity and a photoperiod of 16 h light/8 h dark in a growth chamber. The 3rd-instar VF nymphs of L. striatellus were reared on RBSDV-infected (RB) rice plants for two days. After rearing on healthy rice seedlings for another three days, the nymphs were collected as RB L. striatellus. 2.2. Quantitative Real-Time PCR (RT-qPCR) RT-qPCR was adopted to analyze the relative gene expression as previously de- scribed [26]. Total RNA was extracted by TRIzol reagent (Invitrogen, Waltham, MA, USA). The cDNA was synthesized from 1 µg of total RNA using PrimeScriptTM RT reagent kit Viruses 2021, 13, 1576 3 of 13 with gDNA Eraser (Takara, Kusatsu, Japan) according to the manufacturer’s instruction. Quantitative PCR reactions were conducted on iQ5 real-time PCR system (Bio-Rad, Her- cules, CA, USA) using SYBR PrimeScriptTM RT-PCR Kit (Takara, Kusatsu, Japan). Each PCR reaction included three independent technical and biological replications. Ribosomal protein L5 (RPL5) was used as an internal reference gene [26]. The relative expression levels of target genes were calculated by 2−DDCt [26]. The primers with a product length of 110–160 bp were designed by Beacon Designer 7.7, and the details of the primers are listed in Supplemetary Materials, Table S1. 2.3. RNA Interference (RNAi) For the RNAi assay, partial sequences of CYP6CW1,