Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

Wenjun Zha; Aiqing You

doi:10.1371/journal.pone.0238549

. 2020 Sep 9;15(9):e0238549. doi: 10.1371/journal.pone.0238549

Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

Wenjun Zha ¹, Aiqing You ^1,^2,^*

Editor: Peng He³

PMCID: PMC7480849 PMID: 32903256

Abstract

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a destructive pest that poses a significant threat to rice plants worldwide. To explore how BPHs adapt to the resistant rice variety, we analyzed proteomics profiles of two virulent N. lugens populations. We focused on Biotype Y, which can survive on the moderately resistant rice variety YHY15, and Biotype I, which can survive on the susceptible rice variety TN1. We performed protein quantitation using the isobaric tag for relative and absolute quantification (iTRAQ) and then compared the expression patterns between two virulent N. lugens populations and found 258 differentially expressed proteins (DEPs). We found that 151 of the DEPs were up-regulated, while 107 were down-regulated. We evaluated transcript levels of 8 expressed genes from the iTRAQ results by qRT-PCR, which revealed transcriptional changes that were consistent with the changes at the protein level. The determination of the protein changes in two virulent N. lugens populations would help to better understanding BPH adaptation to resistant rice varieties and facilitate the better design of new control strategies for host defense against BPH.

Introduction

Proteomics is the study of proteins and their impact on cells, tissues and organisms. The data obtained from proteomic analyses provide a macroscopic view of gene expression under different environmental conditions, thereby enabling targeted experiments. Traditionally, proteomic studies based on two-dimensional electrophoresis were used to identify differentially expressed proteins (DEPs), including those related to rapid cold stress response in Lissorhoptrus oryzophilus [1] and immune response in Triatoma dimidiate [2]. With recent technological advances, highly sensitive proteomics techniques have emerged. These new approaches include the isobaric tag for relative and absolute quantification (iTRAQ), which is more reliable than two-dimensional electrophoresis [3]. This high-throughput approach enables DEPs to be incorporated into pathway models [4]. Recent research has shown that iTRAQ can be utilized in many different species, including Laodelphax striatellus [5], Autographa californica [6] and Apis mellifera [7]. A previous study of brown planthopper (Nilaparvata lugens Stål) used an iTRAQ protocol to identify proteins present in the ovary [8].

The brown planthopper is a destructive rice pest that causes severe damage and results in significant yield loss through both direct grain loss as well as transmission of plant viruses [9]. Although insecticides can be used to control damage from BPH and other pests, over usage of them has led to BPH resurgence [10] and has caused environmental problems that threaten human health. Since the first resistant rice variety against BPH was discovered in 1969, more than 30 BPH resistance genes have been reported from different resistance sources [11]. We used a susceptible rice variety (TN1) as a control and a moderately resistant rice variety (YHY15) carrying the resistance gene BPH15 [12]. It has been found that resistance genes impair BPH feeding behavior on varieties and cause BPH physiological changes by increasing mortality rates, extending developmental periods, and reducing reproductive output [10, 13, 14]. BPHs that are allowed to feed on resistant rice for a long time may slowly evolve into new virulent BPH populations to overcome rice resistance [15]. Among different brown planthopper biotypes, the BPH biotype I is widely distributed in East and Southeast Asia and can survive on the TN1 rice variety [16]. The BPH biotype Y is a virulent biotype that has adapted to the moderately resistant rice variety (YHY15) by compelling biotype I BPHs to feed on YHY15 for generations [17]. In both lab and field studies, brown planthopper has been able to adapt to resistant rice rapidly.

In this study, iTRAQ was used to evaluate proteomic differences between two BPH populations, leading to the identification of DEPs, which are correlated with resistance. Among 3167 identified proteins, 258 were considered as differentially expressed in the BPH-YHY15 population relative to the BPH-TN1 population. We then used Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to analyze the functions of these DEPs. Subsequent Clusters of Orthologous Groups of proteins (COG) analysis suggested that a number of those proteins were involved in the regulation of post-translational modification, protein turnover, chaperones pathways. Additional research on these proteins might provide valuable information regarding strategies for BPH management and control.

Materials and methods

Plants and insects

For this study, we utilized TN1 as a susceptible rice variety and YHY15 as a moderately resistant rice variety. Two virulent N. lugens populations (Biotype I and Biotype Y) were maintained on TN1 and YHY15 plants at the Hubei Academy of Agricultural Sciences, Wuhan, China.

Protein extraction

Three different replicates were carried out, using forty adult insects in each. The phenol/methanolic ammonium acetate method was used for protein extraction [18]. The samples were dissolved in extraction media and suspended for 5 s, broken by ultrasonication (2.4 s on, 3 s off) and then incubated at 22°C for 20 min before final centrifugation at 14,000 × g for 25 min at 4°C. The extract containing total insect protein was kept at -80°C for further quantitative proteomic analysis.

iTRAQ labeling and fractionation

Protein digestion was conducted as described previously [18–20]. An aliquot of 200 μg of total protein was collected from each sample and digested with 2 μg trypsin (Promega). Samples were labeled according to the procedure outlined in the 4-plex iTRAQ reagent (Applied Biosystems). Protein samples were labeled as TN1-1:115; YHY15-1:117; TN1-2:115; YHY15-2:117; TN1-3:116; YHY15-3:114, followed by multiplexing and vacuum drying. The labeled samples were collected using a chromatography system and then separated into ten fractions via a substantial cation exchange column.

Nano-LC-MS/MS analysis

Peptides from each fraction were dissolved in solution A (5% ACN, 0.1% FA) and then centrifuged. The supernatant was then fractionated using a RIGOL L-3000RP-HPLC system. Elution was performed by transitioning from 5% solution B (95% ACN, 0.1% FA) to 35% solution B over 40 min, followed by a 5 min linear gradient from 60% up to 80% over 2 min and then 1 min of constant running at 80%. Finally, a 5% solution was used for 1 min and the instrument was equilibrated in solution A. Nano-LC-MS/MS analysis was carried out using a nano-LC equipped with a Q Exactive Mass Spectrometer.

Reference proteins were obtained from the Hemiptera protein database downloaded from the National Center for Biotechnology Information database. Proteins with fold-change values ≥ 1.2 and p-values ≤ 0.05 were considered to be significant DEPs between the two samples.

Bioinformatics analysis

Protein functional annotation of the identified DEPs was carried out using Uniprot. The Clusters of Orthologous Groups of proteins (COG) database information was employed for the functional annotation of DEPs. The Worfpsort and Cello software packages were used to determine the subcellular localization of proteins of interest. Metabolic pathway analysis using the DEPs was performed via the KEGG database [21]. Finally, GO enrichment analysis was carried out to discover enriched pathways [22, 23].

RNA isolation and real-time PCR

Sequences of 8 DEPs identified with iTRAQ were downloaded from the National Center for Biotechnology Information database. Total RNA was isolated from insects using TRIzol (Takara, Japan). cDNA was synthesized using a FastQuant RT Kit (TIANGEN), followed by qRT-PCR via the SuperReal PreMix Plus (Takara, Japan) under the following conditions: 95°C for 2 min; followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 30 s. The qRT-PCR analysis of each sample was carried out three times. Relative expression of each transcript was calculated via the 2^−ΔΔCT method [24] and normalized with ß-Actin, with BPH-TN1 samples used as a negative control.

Results

Quantitative proteomic analysis by iTRAQ labeling

Proteins that were differentially expressed between BPH-YHY15 and BPH-TN1 were evaluated by iTRAQ labeling and quantified by liquid chromatography-mass spectrometry analysis (Fig 1). According to the LC-MS/MS analysis, we identified 3167 proteins in the two populations. Based on the criteria for defining DEPs (fold change ratio ≥ 1.2 and p ≤ 0.05), 151 up-regulated and 107 down-regulated proteins were discovered in the BPH-YHY15 population relative to the BPH-TN1 population (Fig 2). Information on the DEPs and their accession numbers are shown in the S1 Table.

Fig 1 — Differentially expressed proteins were quantified and analyzed by iTRAQ labeling and LC-MS/MS. A bioinformatics analysis was used to explore the relationship between differentially expressed proteins in N. *lugens* and BPH adaptation to rice resistance. qRT-PCR was used to demonstrate that protein abundance changes were correlated with differential expression at the transcriptional level.

Fig 2 — The volcano plot shows up-regulated differentially expressed proteins (red), unchanged proteins (gray), and down-regulated differentially expressed proteins (green).

Functional annotation and classification

DEPs were queried against GO and KEGG databases. We were able to annotate the molecular functions of 293 proteins, including binding proteins and proteins with catalytic activity, transporter activity or structural importance (S2 Table, MF). For cellular components, we annotated 829 proteins present in either the cytosol or organelles (S2 Table, CC). Finally, 1055 proteins were assigned to different biological processes, including cellular processes, single-organism processes and metabolic processes (S2 Table, BP).

Gene Ontology (GO) enrichment analysis of DEPs

We next performed GO enrichment analysis using Fisher's exact test to identify processes that were enriched in the DEPs (S3 Table). Significantly enriched molecular function categories included lipid binding (11 proteins), carbon-carbon lyase activity (4), and alcohol binding (4) (Fig 3A). Cellular components were enriched in extracellular region (49), extracellular space (13), lytic vacuole (9) and lysosome (9) (Fig 3B). Biological process categories included response to chemical (38), response to oxygen-containing compound (20) and regulation of response to stress (19) (Fig 3C). Overall this analysis revealed that the DEPs were enriched in proteins that are responsive to chemicals.

Fig 3 — A: Molecular Function. B: Cellular Component. C: Biological Process. The X-axis represents Fisher's exact test p-value, while the Y-axis represents the name of the pathway. The circle color represents enrichment fold change, and the circle size represents the amount of differentially expressed proteins contained in each category.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

In addition to GO annotation, we also examined the enrichment of DEPs in different KEGG pathways. DEPs were found to be enriched in apoptosis (7 proteins) and lysosome metabolism (7 proteins), suggesting these processes may play critical roles in BPH adaptation to rice resistance (Fig 4, S4 Table). However, further studies of these pathways at the molecular and cellular levels should be performed to corroborate these findings.

Fig 4 — The X-axis represents Fisher's exact test p-value, while the Y-axis represents the name of the pathway. The circle color represents enrichment fold change, and the circle size represents the amount of differentially expressed proteins contained in each category.

Clusters of Orthologous Groups of proteins (COG) pathway enrichment analysis of DEPs

We classified all DEPs with high homology into 25 COG categories (Fig 5; S5 Table). "Post-translational modification, protein turnover, chaperones" (31 proteins) was the most abundant functional category, followed by "general function prediction only" (22 proteins), and "energy production and conversion" (21 proteins). Additionally, many proteins were clustered in groups related to stress response, including "signal transduction mechanisms" (18 proteins), "inorganic ion transport and metabolism" (7 proteins), "transcription" (5 proteins), and "defense mechanisms" (3 proteins). Based on these clusters, we identified 20 DEPs correlated with brown planthopper adaptation to rice resistance from the 258 DEPs, likely lipid transport and metabolism (vitellogenin), secondary metabolites biosynthesis, transport and catabolism (cytochrome P450), amino acid transport and metabolism (serine protease HP21), defense mechanisms (serpin-4), and post-translational modification, protein turnover, chaperones (heat shock protein 70) (Table 1).

Table 1. DEPs associated with the adaptation of brown planthopper to resistant rice.

Accession	Description	Fold change	P-value
BAP87098.1	vitellogenin	2.11	0.00
AGZ04899.1	odorant binding protein 8	1.77	0.01
AGK40915.1	proclotting enzyme-3	1.41	0.00
AID60312.1	serine protease HP21	1.34	0.00
AGK40928.1	serpin-4	1.33	0.01
AJO25056.1	imaginal disc growth factor	1.32	0.00
AIW79986.1	cytochrome P450 CYP6ER1v2	1.31	0.00
AFJ75808.1	glutathione s-transferase M2	1.30	0.00
CAC87119.1	trypsin-like protease	1.21	0.03
AKN21380.1	multicopper oxidase 2	1.20	0.03
AEL88646.1	phosphoacetylglucosamine mutase	0.83	0.00
AID60360.1	trypsin-26	0.82	0.01
AIW79984.1	cytochrome P450 CYP6CS1v2	0.81	0.01
AMZ00358.1	Ras superfamily small GTPase Cdc42	0.81	0.03
AIW79998.1	cytochrome P450 CYP380C10	0.80	0.04
ALP82110.1	beta-tubulin 4	0.78	0.01
AJO25042.1	chitinase, partial	0.76	0.00
CAZ65617.1	cytochrome P450	0.74	0.00
BAI83415.1	sugar transporter 1	0.60	0.00
AFJ20626.1	heat shock protein 70	0.55	0.00

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Comparison of the proteomics data with transcriptional data

To investigate the relationship between the DEPs and their respective transcripts, we performed qRT-PCR on 8 expressed genes from the iTRAQ results (S6 Table), including four up-regulated (vitellogenin, proclotting enzyme-3, serpin-4 and cytochrome P450 CYP6ER1v2) (Fig 6A) and four down-regulated proteins (trypsin-26, beta-tubulin 4, chitinase and sugar transporter 1) (Fig 6B). They were chosen in conformity with the proportion of their up- and downregulation from the iTRAQ results and the availability of the mRNA sequence from the BPH transcriptome. Overall, the differential expression at the transcriptional level was well-correlated with the trends at the protein level, indicating that iTRAQ is a reliable method for differential protein analysis of BPH.

Fig 6 — β-actin was used to normalize gene expression changes. Error bars represent SD, and all analyses were performed in triplicate. * p < 0.05, ** p < 0.01 (Student’s t-test). A: Four genes (*Nl-Vg*, *Nl-proc3*, *Nl-ser4* and *Nl-CYP*) were found to be up-regulated at both the protein and transcriptional levels. B: Four genes (*Nl-try26*, *Nl-tub4*, *Nl-chi* and *Nl-sugt1*) were found to be down-regulated at both levels. Red and green represent the expression levels of BPH-YHY15 by qRT-qPCR and iTRAQ labeled techniques, respectively. CK represents that of BPH-TN1 for negative control.

Discussion

iTRAQ is a new technique that identifies and quantifies proteins by using isobaric reagents to label the primary amines of peptides and proteins in a digestion mixture. S ions are used as the reporter, which enables the quantification of proteins from different sources [25]. This method makes protein assays significantly higher throughput, leading to a significant increase in larger scale protein analyses [26, 27]. To determine the link between altered protein levels and adaptability in brown planthoppers exposed to susceptible and resistant rice varieties, iTRAQ was performed in combination with LC-MS/MS. This led to the identification of 3167 unique proteins, 258 of which were significantly differentially expressed between the two N. lugens populations. We then annotated differentially abundant proteins via GO analysis to determine their putative functions. As expected, several GO terms—including response to a stimulus, response to chemicals, regulation of response to stimulus, response to external stimulus, response to organic substance, cellular response to stress and regulation of cell communication—were found to be substantially enriched. Notably, a large number of the DEPs may be related to response to stimulus.

GO enrichment pathway analysis indicated that response to chemical, response to oxygen-containing compound, and regulation of response to stress related DEPs might be involved in BPH adaptation to rice resistance. Furthermore, KEGG enrichment pathway analysis identified that the DEPs were involved in specific metabolic pathways, including apoptosis metabolism. The result suggested that feeding on moderately resistant plants may induce cell apoptosis in BPH. Apoptosis is an effective means by which a host controls virus infection [28]. Rice ragged stunt virus will induce apoptosis in the salivary gland cells of its insect vector, N. lugens [29]. The apoptosis metabolism may be associated with BPH adaptation to rice resistance, and understanding the nature of these proteins can improve mechanistic understanding of resistance adaptation.

The COG pathway enrichment analysis showed that the largest group (12% of the DEPs) was associated with "post-translational modification, protein turnover, chaperones", such as heat shock protein 70. And we also found several DEPs correlated with brown planthopper adaptation to rice resistance likely lipid transport and metabolism (vitellogenin), secondary metabolites biosynthesis, transport and catabolism (cytochrome P450), and amino acid transport and metabolism (serine protease HP21).

Heat shock 70 kDa proteins (Hsp70) participate in protein folding, as well as protein translocation and caspase-dependent apoptosis [30]. Our results indicated that Hsp70 protein was more abundant in the BPH-YHY15 group, which may increase this group's survival rate at low temperatures. Relatively high levels of hsp70 mRNA are correlated with low temperature tolerance in some insects, such as D. melanogaster [31] and Leptinotarsa decemlineata [32].

Vitellogenin is a precursor protein present in the egg yolk of many oviparous insects. However, insect Vgs are usually synthesized within body fat and processed with many co- and post-translational modifications, including proteolytic cleavage of nascent proteins [33–35]. Previous studies found Vg of Apis mellifera can protect itself from oxidative stress [36]. Moreover, the expression of Nlvg is correlated with the embryonic development stage in N. lugens [37]. In our study, we found that Nlvg expression in BPH-YHY15 individuals was 5.84-fold higher than that of the individuals in the BPH-TN1 population, suggesting that higher lipid deposition ability may be involved in the adaptation response of N. lugens to resistant rice.

Insect detoxification systems often evolve during insect-plant interactions via modification of P450s and glutathione S-transferases that catabolize secondary plant compounds [38–40]. P450s play crucial roles in insect growth and development and are also involved in pesticide metabolism [41]. Several studies have demonstrated that mRNA transcript levels of the BPH P450 gene were induced by some resistant rice varieties [42–44]. Based on our iTRAQ results, we infer that P450 enzymes may be involved in BPH adaptation to the rice YHY15.

Serine proteases are involved in insect digestion and play crucial roles in numerous physiological processes, including larval growth and development [45]. Higher expression levels of serine proteases in host-adapted BPHs suggest that they may offer nutrients required for BPH survival and fecundity.

In summary, iTRAQ led to the detection of 258 DEPs between the BPH-TN1 and BPH-YHY15 groups of BPH. In the DEPs, 151 were up-regulated and 107 were down-regulated. Determining how protein contents differ between host-adapted and non-adapted BPH populations can lead to an improved understanding of the adaptability of BPH to rice resistance at the proteomic level. Moreover, this data may also help us explain other BPH traits, including fecundity and survival. Despite these interesting findings, our study does have some significant limitations. In particular, some DEPs could not be identified and well-quantified, motivating future experimental work on this species. Taken together, our analysis of differences in protein abundance in the two virulent N. lugens populations represents a significant step forward in the understanding of BPH adaptability to resistant rice.

Supporting information

S1 Table. Differentially expressed proteins between BPH-YHY15 and BPH-TN1 samples.

(XLSX)

Click here for additional data file.^{(281.6KB, xlsx)}

S2 Table. Functional annotation and classification of proteins.

(XLSX)

Click here for additional data file.^{(18.2KB, xlsx)}

S3 Table. Functional enrichment in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(32.2KB, xlsx)}

S4 Table. Significantly enriched KEGG pathways in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(10.8KB, xlsx)}

S5 Table. Significantly enriched COG pathways in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(12.6KB, xlsx)}

S6 Table. List of genes selected for qRT-PCR assay.

(XLSX)

Click here for additional data file.^{(9.5KB, xlsx)}

Acknowledgments

We would like to thank TopEdit (www.topeditsci.com) for the English language editing of this manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Financial support was provided by the Major Program of Genetically Modified Organisms Breeding of China (2018ZX08001-01B), the National Key Research and Development Program of China (2018YFD0100806) to WZ, and the Supportive Project of Hubei Academy of Agricultural Sciences (2019fcxjh02) to WZ.

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PLoS One. doi: 10.1371/journal.pone.0238549.r001

Decision Letter 0

Peng He

26 Jun 2020

PONE-D-20-16306

iTRAQ-based quantitative proteomic analysis of proteins associated with the adaptation of brown planthopper to resistant rice varieties

PLOS ONE

Dear Dr. You,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Peng He, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript contributed by Zha et al. investigated related proteins contributed to the adaptation of Nilaparvata lugens to resistant rice varieties by iTRAQ. Authors found differentially expressed proteins (DEPs) between two virulent N. lugens, which were associated with lipid transport and metabolism and defense mechanism. Authors concluded that these DEPs may participate in N. lugens adaptation to resistant rice varieties. However, there are several points or things to be revised or answered.

1. In Short Title, authors should indicate what proteins to be analyzed or what physiological problems to be solved by iTRAQ.

2. The title of this paper is very general, please concretize this title.

3. From line 12 to line 13, authors didn't resolve how the proteins of N. lugens mediate its adaptation to rice resistance throughout the whole paper, only some differentially expressed proteins were identified by iTRAQ. Please revise this sentence expression.

4. From line 14 to line 15, "Biotype Y" and "Biotype I" didn't appear in Materials and Methods. Why were they here? Please explain and revise.

5. From line 19 to line 21, whether the protein expression level can be detected by qRT-PCR? Please make clear this problem.

6. From line 21 to line 23, authors should consider to rewrite this research conclusion. Lipid transport and metabolism and defense mechanism were not showed in KEGG pathway enrichment analysis (3.4), and number of proteins of the two categories is not the most compared with others (3.5). Please explain and revise.

7. In introduction, authors need to introduce reason of selecting YHY15 and TN1.

8. In second paragraph of introduction, the contents of pest resistant rice varieties cultivation and the adaptation mechanism of N. lugens to resistant rice need to be added, which can let readers kown the progress of research about pest resistant rice. Please revise.

9. In third paragraph of introduction, the main research contents of this paper are drafted according to Materials and Methods and Results. Please authors consider to reorganize this part.

10. In Results, the top 20 or 30 differentially expressed proteins (DEPs) between two virulent N. lugens should be considered to be added as table, which can better understand the adaptation mechanism of N. lugens to resistant rice. In this way, this paper can show specific DEPs associated with the adaptation of N. lugens to resistant rice varieties. Please revise.

11. In Discussion, vitellogenin, P450, serine proteases and Hsp70 didn't appear throughout paper Results, why did authors suddenly discuss these proteins? I suggest that discussion is written according to research results. Please reorganize article discussion.

12. In line 258, "Stal" is wrong. Please check similar problem.

Reviewer #2: This manuscript performed protein quantitation using iTRAQ and then compared the expression

patterns between two virulent N. lugens populations and found 258 differentially

expressed proteins. It was well organized and written, thus I recommend acceptance after minor revision.

Major concerns:

1. Two virulent N. lugens populations (BPH-TN1 and BPH-YHY15): should describe basic data of these 2 populations, for example, survival rates on YHY15 or TN1. Alternatively, related references could be cited.

2. 8 DEPs were chosen for qRT-PCR. How to select?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 9;15(9):e0238549. doi: 10.1371/journal.pone.0238549.r002

Author response to Decision Letter 0

18 Jul 2020

Response to Reviewer 1:

Comment 1:

The manuscript contributed by Zha et al. investigated related proteins contributed to the adaptation of Nilaparvata lugens to resistant rice varieties by iTRAQ. Authors found differentially expressed proteins (DEPs) between two virulent N. lugens, which were associated with lipid transport and metabolism and defense mechanism. Authors concluded that these DEPs may participate in N. lugens adaptation to resistant rice varieties. However, there are several points or things to be revised or answered.

1. In Short Title, authors should indicate what proteins to be analyzed or what physiological problems to be solved by iTRAQ.

Answer:

Thanks for the Reviewer’s valuable comments. The short title "iTRAQ-based quantitative proteomic analysis of brown planthopper proteins" was changed to "iTRAQ proteomic reveals the adaptation of brown planthopper to two rice varieties" in the revised manuscript.

Comment 2：

The title of this paper is very general, please concretize this title.

Answer:

Following your suggestion, the title of this paper "iTRAQ-based quantitative proteomic analysis of proteins associated with the adaptation of brown planthopper to resistant rice varieties" was changed to "Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties" in the revised manuscript (Lines 1-2, Page 1).

Comment 3：

From line 12 to line 13, authors didn't resolve how the proteins of N. lugens mediate its adaptation to rice resistance throughout the whole paper, only some differentially expressed proteins were identified by iTRAQ. Please revise this sentence expression.

Answer:

Following your suggestion, we changed " the proteins of N. lugens mediate its adaptation to rice resistance " to " BPHs adapt to the resistant rice variety " in the revised manuscript (Lines 12-13, Page 1).

Comment 4：

From line 14 to line 15, "Biotype Y" and "Biotype I" didn't appear in Materials and Methods. Why were they here? Please explain and revise.

Answer:

We provided a brief description for Biotype Y and Biotype I in the abstract section, because it will help a person outside of this field to comprehend them well. And following your suggestion, we changed "BPH-TN1 and BPH-YHY15" to " Biotype I and Biotype Y " in Materials and Methods section of the revised manuscript (Line 71, Page 2).

Comment 5：

From line 19 to line 21, whether the protein expression level can be detected by qRT-PCR? Please make clear this problem.

Answer:

Following your suggestion, we changed " of these DEPs " to " expressed genes from the iTRAQ results " in the revised manuscript (Line 19, Page 1). And we also changed " selected DEPs " to " expressed genes from the iTRAQ results "(Line 175, Page 5).

Comment 6：

From line 21 to line 23, authors should consider to rewrite this research conclusion. Lipid transport and metabolism and defense mechanism were not showed in KEGG pathway enrichment analysis (3.4), and number of proteins of the two categories is not the most compared with others (3.5). Please explain and revise.

Answer:

Following your suggestion, we changed " Our evidence suggests that DEPs of N. lugens are associated with lipid transport and metabolism, as well as defense mechanisms, which may contribute to BPH adaptation to resistant rice varieties. " to " The determination of the protein changes in two virulent N. lugens populations would help to better understanding BPH adaptation to resistant rice varieties and facilitate better design of new control strategies for host defense against BPH. " in the revised manuscript (Lines 21-23, Page 1).

Comment 7：

In introduction, authors need to introduce reason of selecting YHY15 and TN1.

Answer:

Following your suggestion, we added the reason why these 2 populations were chosen. We added the paragraph “Among different brown planthopper biotypes, the BPH biotype I is widely distributed in East and Southeast Asia and can survive on the TN1 rice variety [16]. The BPH biotype Y is a virulent biotype that has adapted to the moderately resistant rice variety (YHY15) by compelling biotype I BPHs to feed on YHY15 for generations [17]. "into our revised manuscript and the details can be found in Lines 51-55, Page 2.

Comment 8：

In second paragraph of introduction, the contents of pest resistant rice varieties cultivation and the adaptation mechanism of N. lugens to resistant rice need to be added, which can let readers kown the progress of research about pest resistant rice. Please revise.

Answer:

Following your suggestion, we added the contents of pest resistant rice varieties cultivation and the adaptation mechanism of N. lugens to resistant rice. We added the paragraph “Since the first resistant rice variety against BPH was discovered in 1969, more than 30 BPH resistance genes have been reported from different resistance sources [11]. We used a susceptible rice variety (TN1) as a control and a moderately resistant rice variety (YHY15) carrying the resistance gene BPH15 [12]. It has been found that resistance genes impair BPH feeding behavior on varieties and cause BPH physiological changes by increasing mortality rates, extending developmental periods, and reducing reproductive output [10, 13, 14]. BPHs that are allowed to feed on resistant rice for a long time may slowly evolve into new virulent BPH populations to overcome rice resistance [15]. " into our revised manuscript and the details can be found in Lines 44-51, Page 2.

Comment 9：

In third paragraph of introduction, the main research contents of this paper are drafted according to Materials and Methods and Results. Please authors consider to reorganize this part.

Answer:

Following your suggestion, we reorganized this part according to Results. The paragraph is “In this study, iTRAQ was used to evaluate proteomic differences between two BPH populations, leading to the identification of DEPs which are correlated with resistance. Among 3167 identified proteins, 258 were considered as differentially expressed in the BPH-YHY15 population relative to the BPH-TN1 population. We then used Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to analyze the functions of these DEPs. Subsequent Clusters of Orthologous Groups of proteins (COG) analysis suggested that a number of those proteins were involved in the regulation of post-translational modification, protein turnover, chaperones pathways. Additional research on these proteins might provide valuable information regarding strategies for BPH management and control.” in the revised manuscript (Lines 57-66, Page 2).

Comment 10：

In Results, the top 20 or 30 differentially expressed proteins (DEPs) between two virulent N. lugens should be considered to be added as table, which can better understand the adaptation mechanism of N. lugens to resistant rice. In this way, this paper can show specific DEPs associated with the adaptation of N. lugens to resistant rice varieties. Please revise.

Answer:

Following your suggestion, we added specific DEPs associated with the adaptation of N. lugens to resistant rice varieties in Table 1 of our revised manuscript. And information on the DEPs and their accession numbers are shown in S1 Table.

Comment 11：

In Discussion, vitellogenin, P450, serine proteases and Hsp70 didn't appear throughout paper Results, why did authors suddenly discuss these proteins? I suggest that discussion is written according to research results. Please reorganize article discussion.

Answer:

Following your suggestion, we added vitellogenin, P450, serine proteases and Hsp70 into the results of our revised manuscript (Lines 166-171, Page 5). The paragraph is “Based on these clusters, we identified 20 DEPs correlated with brown planthopper adaptation to rice resistance from the 258 DEPs, likely Lipid transport and metabolism (vitellogenin), Secondary metabolites biosynthesis, transport and catabolism (cytochrome P450), Amino acid transport and metabolism (serine protease HP21), Defense mechanisms (serpin-4), and Posttranslational modification, protein turnover, chaperones (heat shock protein 70) （Table 1）.”

Comment 12：

In line 258, "Stal" is wrong. Please check similar problem.

Answer:

Following your suggestion, we have checked all the similar problems.

Response to Reviewer 2:

Comment 1：

Two virulent N. lugens populations (BPH-TN1 and BPH-YHY15): should describe basic data of these 2 populations, for example, survival rates on YHY15 or TN1. Alternatively, related references could be cited.

Answer:

Following your suggestion, we added the reason why these 2 populations were chosen. We added the paragraph " Since the first resistant rice variety against BPH was discovered in 1969, more than 30 BPH resistance genes have been reported from different resistance sources [11]. We used a susceptible rice variety (TN1) as a control and a moderately resistant rice variety (YHY15) carrying the resistance gene BPH15 [12]. It has been found that resistance genes impair BPH feeding behavior on varieties and cause BPH physiological changes by increasing mortality rates, extending developmental periods, and reducing reproductive output [10, 13, 14]. BPHs that are allowed to feed on resistant rice for a long time may slowly evolve into new virulent BPH populations to overcome rice resistance [15]. Among different brown planthopper biotypes, the BPH biotype I is widely distributed in East and Southeast Asia and can survive on the TN1 rice variety [16]. The BPH biotype Y is a virulent biotype that has adapted to the moderately resistant rice variety (YHY15) by compelling biotype I BPHs to feed on YHY15 for generations [17]. " into our revised manuscript and the details can be found in Lines 44-55, Page 2.

Comment 2：

8 DEPs were chosen for qRT-PCR. How to select?

Answer:

Following your suggestion, we added the reason why 8 DEPs were chosen for qRT-PCR. We added the sentence" They were chosen in conformity with the proportion of their up- and downregulation from the iTRAQ results and the availability of the mRNA sequence from the BPH transcriptome." into our revised manuscript and the details can be found in Lines 178-180, Page 5.

PLoS One. doi: 10.1371/journal.pone.0238549.r003

Decision Letter 1

Peng He

6 Aug 2020

PONE-D-20-16306R1

Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

PLOS ONE

Dear Dr. You,

Please submit your revised manuscript by Sep 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Peng He, Ph.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The manuscript contributed by Zha et al. characterized proteomic profiling of proteins associated with the adaptation of brown planthopper to resistant rice varieties by iTRAQ. Authors found differentially expressed proteins (DEPs) between two virulent N. lugens, which would help to better understanding BPH adaptation to resistant rice varieties and facilitate better design of new control strategies for host defense against BPH. Although authors revised several points according to the academic editor and reviewers suggestions, there are several points or things to be revised or answered.

1. In Keywords section, the selection of keywords should be consistent with professional research content, please add keyword.

2. The quality of the written English requires improvement for better.

(1) In line 80, a space is required in "-80°C". Please check for similar problems in this paper.

(2) In line 93, a space is required in "5%ACN". Please check for similar problems in this paper.

(3) "Nano-LC-MS/MS" of line 92 and "Nano LC-MS/MS" of line 98 are not consistent.

(4) Please revise font size in line 166-171. Please check for similar problems in the whole paper.

3. Please add statistical analysis in Figure 6.

4. In Discussion, authors should add some discussion contents about GO enrichment analysis of DEPs, KEGG pathway enrichment analysis of DEPs, COG pathway of DEPs. Vitellogenin, P450, serine proteases and Hsp70 should be integrated into pathway to discuss, Please consider whether any changes are needed. In addition, reference 36 and 37 are about insecticide resistance, which are not suitable here, please revise.

Reviewer #2: The authors addressed all comments raised by the reviewers, so I have no more questions. Thus, I suggest it be accepted for publication in the journal.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Sep 9;15(9):e0238549. doi: 10.1371/journal.pone.0238549.r004

Author response to Decision Letter 1

16 Aug 2020

Response to Reviewer 1:

Comment 1:

The manuscript contributed by Zha et al. characterized the proteomic profiling of proteins associated with the adaptation of brown planthopper to resistant rice varieties by iTRAQ. Authors found differentially expressed proteins (DEPs) between two virulent N. lugens, which would help to better understanding BPH adaptation to resistant rice varieties and facilitate the better design of new control strategies for host defense against BPH. Although authors revised several points according to the academic editor and reviewers suggestions, there are several points or things to be revised or answered.

1. In the Keywords section, the selection of keywords should be consistent with professional research content, please add the keyword.

Answer:

Thanks for the Reviewer’s valuable comments. We added the keywords "adaptation; GO analysis; KEGG pathway analysis; COG pathway analysis" into the revised manuscript.

Comment 2：

The quality of written English requires improvement for better.

(1) In line 80, a space is required in "-80°C". Please check for similar problems in this paper.

Answer:

Following your suggestion, "-80°C" was changed to "-80 °C" in the revised manuscript (Line 80, Page 2). And we have checked all the similar problems.

(2) In line 93, a space is required in "5%ACN". Please check for similar problems in this paper.

Answer:

Following your suggestion, a space was added into "5%ACN" in the revised manuscript (Line 93, Page 3).

(3) "Nano-LC-MS/MS" of line 92 and "Nano LC-MS/MS" of line 98 are not consistent.

Answer:

Following your suggestion, "Nano LC-MS/MS" was changed to "Nano-LC-MS/MS" in the revised manuscript (Line 98, Page 3).

(4) Please revise the font size in lines 166-171. Please check for similar problems in the whole paper.

Answer:

Following your suggestion, we revised the font size in lines 166-171, Page 5.

Comment 3：

Please add statistical analysis in Figure 6.

Answer:

Following your suggestion, we added the statistical analysis (* p < 0.05, ** p < 0.01) into the Figure 6.

Comment 4：

In Discussion, authors should add some discussion contents about GO enrichment analysis of DEPs, KEGG pathway enrichment analysis of DEPs, COG pathway of DEPs. Vitellogenin, P450, serine proteases and Hsp70 should be integrated into the pathway to discuss. Please consider whether any changes are needed. In addition, reference 36 and 37 are about insecticide resistance, which are not suitable here, please revise.

Answer:

Following your suggestion, first, we added some discussion contents about GO enrichment analysis of DEPs, KEGG pathway enrichment analysis of DEPs, COG pathway of DEPs in the revised manuscript. The paragraph is, “GO enrichment analysis indicated that response to chemical, response to oxygen-containing compound, and regulation of response to stress related DEPs might be involved in BPH adaptation to rice resistance. Furthermore, KEGG pathway enrichment analysis identified that the DEPs were involved in specific metabolic pathways, including apoptosis metabolism. The result suggested that feeding on moderately resistant plants may induce cell apoptosis in BPH. Apoptosis is an effective means by which a host controls virus infection [28]. Rice ragged stunt virus will induce apoptosis in the salivary gland cells of its insect vector, N. lugens [29]. The apoptosis metabolism may be associated with BPH adaptation to rice resistance, and understanding the nature of these proteins can improve mechanistic understanding of resistance adaptation.COG pathway enrichment analysis showed that the largest group (12% of the DEPs) was associated with "post-translational modification, protein turnover, chaperones", such as heat shock protein 70. And we also found several DEPs correlated with brown planthopper adaptation to rice resistance, likely lipid transport and metabolism (vitellogenin), secondary metabolites biosynthesis, transport and catabolism (cytochrome P450), and amino acid transport and metabolism (serine protease HP21).” (Lines 199-214, Pages 5-6).

Second, vitellogenin, P450, serine proteases and hsp70 were integrated into the COG pathway in the discussion part of the revised manuscript.

Last, we deleted the reference 36 and 37 in the revised manuscript.

PLoS One. doi: 10.1371/journal.pone.0238549.r005

Decision Letter 2

Peng He

19 Aug 2020

Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

PONE-D-20-16306R2

Dear Dr. You,

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PLoS One. doi: 10.1371/journal.pone.0238549.r006

Acceptance letter

Peng He

24 Aug 2020

PONE-D-20-16306R2

Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

Dear Dr. You:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Differentially expressed proteins between BPH-YHY15 and BPH-TN1 samples.

(XLSX)

Click here for additional data file.^{(281.6KB, xlsx)}

S2 Table. Functional annotation and classification of proteins.

(XLSX)

Click here for additional data file.^{(18.2KB, xlsx)}

S3 Table. Functional enrichment in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(32.2KB, xlsx)}

S4 Table. Significantly enriched KEGG pathways in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(10.8KB, xlsx)}

S5 Table. Significantly enriched COG pathways in differentially expressed proteins.

(XLSX)

Click here for additional data file.^{(12.6KB, xlsx)}

S6 Table. List of genes selected for qRT-PCR assay.

(XLSX)

Click here for additional data file.^{(9.5KB, xlsx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Comparative iTRAQ proteomic profiling of proteins associated with the adaptation of brown planthopper to moderately resistant vs. susceptible rice varieties

Wenjun Zha

Aiqing You

Roles

Abstract

Introduction

Materials and methods

Plants and insects

Protein extraction

iTRAQ labeling and fractionation

Nano-LC-MS/MS analysis

Bioinformatics analysis

RNA isolation and real-time PCR

Results

Quantitative proteomic analysis by iTRAQ labeling

Fig 1. Overview of the experimental workflow.

Fig 2. Volcano plots of iTRAQ-labeled proteins.

Functional annotation and classification

Gene Ontology (GO) enrichment analysis of DEPs

Fig 3. Gene Ontology enrichment analysis of differentially expressed proteins identified in BPH-YHY15 and BPH-TN1.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

Fig 4. Scatter diagram of enriched Kyoto Encyclopedia of Genes and Genomes pathways.

Clusters of Orthologous Groups of proteins (COG) pathway enrichment analysis of DEPs

Fig 5. Clusters of orthologous groups of proteins pathway enrichment analysis of differentially expressed proteins.

Table 1. DEPs associated with the adaptation of brown planthopper to resistant rice.

Comparison of the proteomics data with transcriptional data

Fig 6. Correlation between iTRAQ results and qRT-PCR results.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Peng He

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Author response to Decision Letter 0

Decision Letter 1

Peng He

Roles

Author response to Decision Letter 1

Decision Letter 2

Peng He

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Acceptance letter

Peng He

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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