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Functional Analysis of Nuclear Factor Y in the Wing-Dimorphic Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with the ability to bind to a CCAAT box in nearly all eukaryotes. However, the function of NF-Y in the life-history traits of insects is unclear. Here, we identified three NF-Y subunits, NlNF-YA, NlNF-YB, and NlNF-YC, in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens. Spatio-temporal analysis indicated that NlNF-YA, NlNF-YB, and NlNF-YC distributed extensively in various body parts of fourth-instar nymphs, and were highly expressed at the egg stage. RNA interference (RNAi)-mediated silencing showed that knockdown of NlNF-YA, NlNF-YB, or NlNF-YC in third-instar nymphs significantly extended the fifth-instar duration, and decreased nymph-adult molting rate. The addition of 20-hydroxyecdysone could specifically rescue the defect in adult molting caused by NlNF-YARNAi, indicating that NlNF-Y might modulate the ecdysone signaling pathway in the BPH. In addition, NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi led to small and moderately malformed forewings and hindwings, and impaired the normal assembly of indirect flight muscles. Adult BPHs treated with NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi produced fewer eggs, and eggs laid by these BPHs had arrested embryogenesis. These findings deepen our understanding of NF-Y function in hemipteran insects.


INTRODUCTION
Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor that exists in almost all organisms, from prokaryotes to eukaryotes (Dorn et al., 1987;Laloum et al., 2013). NF-Y exerts differential regulation on a wide variety of genes through binding to a CCAAT box, one of the most ubiquitous elements in eukaryotic promoters (Li et al., 1992;Mantovani, 1998;Suzuki et al., 2001). NF-Y consists of three subunits, NF-YA, NF-YB, and NF-YC, all required for DNAbinding (McNabb et al., 1995;Sinha et al., 1995;Bi et al., 1997;Mantovani, 1999). Accumulated evidence indicates that animal NF-Y is essential for numerous biological processes involved in proliferation and apoptosis, cancer and tumorigenesis, stress responses, growth, and development (Li et al., 2018). For instance, the deletion of the mouse NF-YA homolog causes early embryo lethality (Bhattacharya et al., 2003). Down-regulation of mouse NF-YA was found to reduce the expression of several cell cycle control genes in differentiated muscle cells, suggesting NF-YA is involved in specifying myogenic fate (Gurtner et al., 2003). Knockdown or overexpression of the Drosophila melanogaster NF-YA homolog in all tissues results in lethality at the larval stage, indicating that a certain level of NF-YA is necessary for viability in flies (Yoshioka et al., 2007). However, he contribution of NF-Y homologs to the life-history traits of other insect species has yet to be reported.
The wing-dimorphic brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is the most destructive pest of rice in Asia (Xue et al., 2014), causing a loss in rice production of more than $300 million annually in Asia. It feeds exclusively on the phloem sap of the rice plant and transmits plant viruses such as rice ragged stunt virus and rice grassy stunt virus, resulting in loss of plant vigor and reduced yield (Backus et al., 2005;Otuka, 2013). The BPH is a hemimetabolous insect, the newly hatched first-instar nymphs look like miniature adults except for the absence of wings and sexual immaturity, and grow gradually within increasing stages. The BPH has five nymphal stages, and wing buds grow with each of these stages, but short-and long-winged morphs are externally indistinguishable until the adults emerge (Xu and Zhang, 2017). Long-winged BPH adults have well-developed forewings, hindwings, and indirect flight muscles (IFM), whereas, short-winged adults have undeveloped forewings and rudimentary hindwings (Xu and Zhang, 2017;Zhang et al., 2019). Wing dimorph and high fecundity are the two most important biological features contributing to the ecological success of the BPH (Zhang et al., 2019). Recently, facilitated by the development of genetic tools Xue et al., 2018) and genomic information (Xue et al., 2014), the molecular basis underlying wing dimorphism (Xu et al., 2015) and population resurgence (Wu et al., 2020) are emerging, which offers a basis for the design of new control agents to combat BPH infestation.
In the present study, we identified the BPH NF-YA (NlNF-YA), NF-YB (NlNF-YB), and NF-YC (NlNF-YC) homologs by searching the BPH genomic and transcriptomic databases. RNA interference (RNAi)-mediated gene silencing showed that NlNF-YA, NlNF-YB, and NlNF-YC all played pivotal roles in nymph growth, wing formation, IFM development, and reproduction. In addition, we found that NF-Y regulated adult eclosion most likely through linking to the ecdysone signaling pathway. Our findings deepen our understanding of the function of NF-Y in hemipteran insects.

Insects
The BPH strain was originally collected from a rice field in Hangzhou, China. Insects were reared on rice seedlings (rice variety: Xiushui 134) in a walk-in chamber at 26 ± 0.5 • C, with a light: dark photoperiod of 16: 8 h and relative humidity of 50 ± 5%.

Gene Identification and Sequence Analysis
The amino acid sequences of Drosophila NF-Y homologs were used to screen BPH genomic and transcriptomic databases for homologs. Total RNA was isolated from BPH nymphs using RNAiso plus (Takara #9109) according to the manufacturer's protocol. For cDNA syntheses, 450 ng of total RNA was reversely transcribed in a 10 µL reaction with HiScript QRT SuperMix (Vazyme #R223-01). The NlNF-YA, NlNF-YB, and NlNF-YC sequences were amplified from cDNA using fidelity DNA polymerase (Toyobo #930700) with NlNF-YA-F/R, NlNF-YB-F/R, and NlNF-YC-F/R primer pairs, respectively (Supplementary Table 1). The PCR product was cloned and the sequence was determined by Sanger sequencing.

RNAi and Microinjection
The dsRNAs were synthesized using a T7 High Yield Transcription Kit (Vazyme #TR101-02) according to the manufacturer's instructions with primers containing the T7 RNA polymerase promoter at both ends (Supplementary Table 1). A dsRNA injection was carried out as in our previous study (Xu et al., 2015). Briefly, fourth-instar nymphs were anesthetized with carbon dioxide for 10-15 s. Approximately 150 ng dsRNA was microinjected into the mesothorax using a FemtoJet microinjection system (Eppendorf). To investigate whether disruption of NlNF-YA affected ecdysone signaling activity, fifth-instar nymphs (n = 10 for each of three replicates) were collected to examine the expression levels of NlE74A and NlE75B by qPCR using corresponding primers (Supplementary Table 1). To investigate RNAi efficiency, BPHs (n = 5 for each of three replicates) at 24 h after adult eclosion were collected for qPCR assay.

Microinjection With 20-hydroxyecdysone (20E)
Fourth-instar nymphs (36-48 hAE) were microinjected with corresponding dsRNAs targeting each gene, and then nymphs were maintained on fresh rice seedings until the fifth-instar stage (within 48 h). Fifth-instar nymphs were microinjected with 20E (1 mg/ml) or distilled water, and the adult eclosion rate was calculated.

Transmission Electron Microscopy
Fourth-instar nymphs (36-48 hAE) were microinjected with dsRNAs targeting NlNF-YA, NlNF-YB, NlNF-YC, or GFP. The thoraxes were dissected from adult females (24 h after emergence) for Transmission Electron Microscopy (TEM) as in our previous study . Briefly, samples were fixed in 2.5% glutaraldehyde overnight at 4 • C. After fixation, samples were post-fixed in 1% osmium tetroxide for 1.5 h. Then, samples were dehydrated in a standard ethanol/acetone series, infiltrated and embedded in Spurr medium, and then superthin sections were cut. The sections were stained with 5% uranyl acetate followed by Reynolds' lead citrate solution and observed under a JEM-1230 transmission electron microscope (JEOL).

Fecundity Assay
Newly emerged females and males (within 12 h after eclosion) were microinjected with dsNF-YA, ds-NF-YB, dsNF-YC, or dsGfp. Then each female was allowed to match with two males in a glass tube. Insects were removed at 5 days after matching, and eggs were counted under a Leica S8AP0 stereomicroscope.

Data Analysis
Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). Means were compared using a two-tailed Student's t-test and log-rank (Mantel-Cox) test at a significance level set at * P < 0.05, * * P ≤ 0.01, and * * * P ≤ 0.001.

Knockdown of NlNF-Y Genes Leads to Defective IFM
Because short-wing BPHs lack hindwings and IFM, we used long-wing BPHs to investigate whether NlNF-YA, NlNF-YB, and NlNF-YC had any effect on IFM development. For this,  . Bars represent the mean ± s.d. derived from independent biological replicates (n = 20). Statistical comparisons between two groups were performed using a two-tailed Student's t-test (n.s., non-significant).
Frontiers in Genetics | www.frontiersin.org fourth-instar long-wing-destined nymphs were microinjected with corresponding dsRNAs targeting each gene. NlNF-YA RNAi , NlNF-YB RNAi , and NlNF-YC RNAi gave rise to adults with curved forewings and hindwings ( Figure 5A). In line with the phenotype in short-winged BPHs, smaller forewings and hindwings were observed in adults previously treated with either NlNF-YA RNAi , NlNF-YB RNAi , or NlNF-YC RNAi (Figure 4B), although there was no discernable effect on hind tibia length ( Figure 4C). Next, these adults were collected for IFM ultrastructure examination using TEM. In GFP RNAi adults, sarcomeres were clearly divided by Z discs and well-organized (Figure 6). In contrast, NlNF-YA RNAi , NlNF-YB RNAi , or NlNF-YC RNAi led to defective sarcomeres, with deformed Z discs and weakly organized myofibrillar. These events indicated that the NlNF-Y complex might be essential for IFM assembly in BPH.

Knockdown of NlNF-Y Genes Impairs Nymphal Growth and Adult Molting
Given that NlNF-Y genes were evenly expressed across nymphal stages (Figure 2A), we investigated whether knockdown of NlNF-Y genes would affect nymph growth. For this purpose, third-instar nymphs were microinjected with corresponding dsRNAs targeting each gene. Nymphs treated with NlNF-YA RNAi , NlNF-YB RNAi , or NlNF-YC RNAi had an extended fifthinstar duration compared with those treated with GFP RNAi (Figure 7A), with NlNF-YA RNAi showing the most profound effect (Figure 7A). In addition, the majority of NlNF-YA RNAitreated nymphs died before adult emergence (Figure 7B), indicating that the disruption of the NF-Y complex impaired nymph-to-adult ecdysis.
Given that ecdysone is a hormone that initiates all major developmental transitions from the egg, to larva, to pupa, to adult in insects (Gilbert et al., 2002;Dubrovsky, 2005), we asked whether it mediated the effects of NlNF-YA on adult molting. For this purpose, we first assessed the expression levels of two early ecdysone response genes, NlE74A and NlE75E (Dubrovsky, 2005), in the context of NlNF-YA knockdown. NlNF-YA RNAi significantly reduced both NlE74A and NlE75E transcripts compared with GFP RNAi (Figure 7C), indicating that knockdown of NlNF-YA might impair ecdyson signaling activity. Following this observation, we asked whether the addition Bar represents mean ± s.d. derived from independent biological replicates (n = 20). Statistical comparisons between two groups were performed using a two-tailed Student's t-test (n.s., non-significant). of ecdysone could rescue the NlNF-YA RNAi defect. To this end, fourth-instar nymphs were microinjected with dsNlNF-YA, followed by microinjection with 20E when nymphs proceeded to the fifth-instar at 48 h after eclosion. The addition of 20E could partially restore the molting defect caused by NlNF-YA RNAi ( Figure 7B) compared with the addition of water, indicating that NlNF-YA RNAi decreased adult eclosion at least partially through the ecdysone signaling pathway.

Knockdown of NlNF-Y Genes Impairs Reproduction
Given that relatively high amounts of NlNF-YA, NlNF-YB, and NlNF-YC transcripts were detected at the early egg stage (Figure 2A), we asked whether NlNF-YA, NlNF-YB, and NlNF-YC contributed to BPH reproduction. Female and male adults at 12 h after eclosion were microinjected with corresponding dsRNAs targeting each gene, and then allowed to mate and to lay eggs for 5 days. BPHs treated with NlNF-YA RNAi , NlNF-YB RNAi , and NlNF-YC RNAi laid substantially fewer eggs than those treated with GFP RNAi (Figure 8A). In addition, the eggs laid by BPHs previously treated with NlNF-YA RNAi , NlNF-YB RNAi , or NlNF-YC RNAi failed to develop eye pigmentation, a characteristic hallmark of egg development, indicating NlNF-Y genes might be essential for embryogenesis ( Figure 8B).

DISCUSSION
In this study, we investigated the functions of individual NlNF-Y members in the BPH during nymphal development, adult molting, wing growth, IFM development, and reproduction. The results demonstrated that NlNF-Y played pivotal roles in these processes. Depletion of NlNF-YA, NlNF-YB, and NlNF-YC resulted in extended nymphal duration, affected nymph-adult molting progress, reduced wing size, disrupted IFM assembly, reduced egg production, and impaired egg development. These findings provide an impetus to understand the function of NF-Y in life-history traits of insects.
The CCAAT box is one of the most common cis-acting elements found in the promoter and enhancer regions of various genes in eukaryotes (Li et al., 1992). An analysis of a large database of 1,031 human promoters indicated that the CCAAT box is present in 63% of them (Suzuki et al., 2001). NF-Y is the major CCAAT box recognizing protein that binds to DNA with high specificity and affinity (Bi et al., 1997;Mantovani, 1998). Knockdown or overexpression of the Drosophila NF-YA homolog with different GAL4 also drives lethality in the Drosophila pharate adult stage, possibly by influencing disc specification (Yoshioka et al., 2007;Ly et al., 2013). A similar phenomenon was observed in BPH, The nymph-adult eclosion rate of BPHs. Fourth-instar nymphs (36-48 hAE) were microinjected with dsRNAs targeting NlNF-YA or Gfp, and then 20E or distilled water was microinjected when nymphs proceeded the fifth-instar stage. (C) The expression of NlE74A and NlE75B in context of NlNF-YA knockdown. The relative expressions of NlE74A and NlE75B were normalized to the expression of rps11. Statistical comparisons between two groups were performed using two-tailed Student's t-test (*P < 0.05, and **P < 0.01).
FIGURE 8 | Fecundity of adults with gene knockdown and phenotype of eggs produced. (A) Newly emerged adults (0-12 hAE) were treated with dsRNAs targeting each gene. The dsRNA-treated females and males were allowed to mate and laid eggs for 5 days. Each circle represents eggs produced by an individual female (n = 6). Bars represent the mean ± s.d. Statistical comparisons between two groups were performed using a two-tailed Student's t-test (***P < 0.001).
(B) Morphology of eggs deposited after 5 days by dsRNA-treated BPHs. The eye pigmentation is indicated by an arrow head.
depleting NlNF-YA, NlNF-YB, and NlNF-YC delayed fifth-instar development and led to high lethality, indicating that the CCAAT box might also be a common cis-acting element in the BPH. In addition, we noticed that depletion either NlNF-YA, NlNF-YB, or NlNF-YC led to small and malformed forewings and hindwings. As a wing-dimorphic insect, fifth-instar BPH nymphs have the ability to develop into either short-winged or longwinged adults. A recent finding showed that cells of short wing pads are largely in the G2/M phase of the cell cycle, whereas those of long wings are largely in G1, indicating that cell cycle progression is necessary for wing morph determination (Lin et al., 2020). These observations are in line with previous reports that cell cycle-related genes are the main target genes of NF-Y (Zwicker et al., 1995;Bolognese et al., 1999;Farina et al., 1999;Kao et al., 1999;Korner et al., 2001;Manni et al., 2001). In addition, the depletion of NlNF-Y not only affected wing size, but also led to an IFM defect, the latter tissue is only present in long-winged adults. Based on these events, we speculate that NF-Y might be tightly involved in regulating alternative wing morphs in the BPH although this needs to be further confirmed experimentally.
It is of interest that the depletion of NlNF-YA significantly decreased the adult molting rate, and this defect could be partially rescued by the addition of 20E. In addition, the depletion of NlNF-YA significantly reduced the expression levels of NlE74A and NlE75B, the downstream genes of the ecdysone pathway. These findings indicated that NlNF-Y might affect the ecdysone pathway although the underlying mechanism remains unknown.
Although there remains much to be done, our findings provided a first glimpse of the function of NF-Y in hemipteran insects.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
H-HC and H-JX designed the experiment and wrote the manuscript. H-HC, Y-LL, X-YL, and J-LZ conducted the experiments. H-JX managed and directed the project. All authors contributed to the article and approved the submitted version.