A mucin-like protein of planthopper is required for ...

Plant Physiology Preview. Published on November 13, 2017, as DOI:10.1104/pp.17.00755

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Short title: Planthopper MLP induces plant immunity response

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Correspondence: Guangcun He, National Key Laboratory of Hybrid Rice, College of

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Life Sciences, Wuhan University, Wuhan 430072, China

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E-mail: [email protected]

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Tel: +86-27-68752384

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A mucin-like protein of planthopper is required for feeding and induces immunity

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response in plants

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Xinxin Shangguan1, Jing Zhang1, Bingfang Liu1, Yan Zhao1, Huiying Wang1,

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Zhizheng Wang1, Jianping Guo1, Weiwei Rao1, Shengli Jing1, Wei Guan1, Yinhua Ma1,

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Yan Wu1, Liang Hu1, Rongzhi Chen1, Bo Du1, Lili Zhu1, Dazhao Yu2 & Guangcun

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He1*

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430072 Wuhan, China; 2Institute for Plant Protection and Soil Sciences, Hubei

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Academy of Agricultural Sciences, 430064 Wuhan, China.

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University,

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One sentence Summary: A secreted mucin-like protein in the rice brown planthopper (Nilaparvata lugens) enables insect feeding and induces plant immune responses.

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Footnotes:

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Author contributions: G.H. conceived the original research plans and supervised the

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experiments. G.H. and X.S. designed the experiments. X.S. carried out most of the

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experiments. J.Z., B.L., H.W., Z.W., S.J., W.G., R.C., B.D., L.Z., and D.Y. carried out

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some of the experiments. G.H. and X.S. analyzed data and wrote the manuscript.

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Funding information: This work was supported by grants from National Program on

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Research & Development of Transgenic Plants Grants (2016ZX08009003-001-008),

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National Natural Science Foundation of China (31630063, 31230060, 31401732), and

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National

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2016YFD0100900).

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Corresponding author: Guangcun He ([email protected])

Key

Research

and

Development

Program

(2016YFD0100600,

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Downloaded from on February 6, 2018 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Copyright 2017 by the American Society of Plant Biologists

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Glossary

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BPH: brown planthopper;

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ET: ethylene;

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GO: gene ontology;

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COG: clusters of orthologous groups;

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dsRNA: double-stranded RNA;

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FDA: fluorescein diacetate;

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GFP: green fluorescent protein;

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HR: hypersensitive response;

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IIM: Intestinal mucins;

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LUC: luciferase;

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JA: jasmonic acid;

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MAPK: mitogen-activated protein kinase;

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NlMLP: N. lugens-secreted mucin-like protein;

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ORF: open reading frame;

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PAMPs: pathogen-associated molecular patterns;

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PRR: Pattern recognition receptor

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PTI: PAMP-triggered immunity

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RLUC: Renilla luciferase gene

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RNAi: RNA interference;

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qRT-PCR: quantitative reverse-transcription PCR;

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SA: salicylic acid;

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SEM: scanning electron microscopy;

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SHP: structural sheath protein;

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SIPK: SA-induced protein kinase;

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Ubi: ubiquitin;

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VIGS: virus-induced gene silencing;

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WIPK: wounding-induced protein kinase

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Abstract

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The brown planthopper, Nilaparvata lugens (Stål), is a pest that threatens rice

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production worldwide. While feeding on rice plants, planthoppers secrete saliva, which

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plays crucial roles in nutrient ingestion and modulating plant defense responses,

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although the specific functions of salivary proteins remain largely unknown. We

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identified a N. lugens-secreted mucin-like protein (NlMLP) by transcriptome and

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proteome analyses and characterized its function, both in brown planthopper and in

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plants. NlMLP is highly expressed in salivary glands and is secreted into rice during

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feeding. Inhibition of NlMLP expression in planthoppers disturbs the formation of

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salivary sheaths, thereby reducing their performance. In plants, NlMLP induces cell

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death, the expression of defense-related genes, and callose deposition. These defense

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responses are related to Ca2+ mobilization and the MEK2 MAP kinase and JA

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signaling pathways. The active region of NlMLP that elicits plant responses is located

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in its C-terminus. Our work provides a detailed characterization of a salivary protein

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from a piercing-sucking insect other than aphids. Our findings that the protein that

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functions in plant immune responses offer new insights into the mechanism

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underlying interactions between plants and herbivorous insects.

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Introduction

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Plants are subjected to attack by diverse herbivorous insects, which are generally

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classified based on their feeding strategies as chewing or piercing-sucking insects.

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Chewing insects, such as caterpillars and beetles, can cause serious mechanical damage

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to plant tissues, whereas piercing-sucking insects feed on plants through specially

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adapted mouthparts known as stylets and cause only limited physical damage to plant

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tissues (Walling, 2000). Insects can also injure plants indirectly by transmitting viral,

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bacterial, and fungal pathogens. Plants use sophisticated perception systems to detect

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insect feeding through cues derived not only from damage caused by feeding

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(Reymond et al., 2000), but also from insect saliva, oral secretions, eggs, volatiles, and

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microbes associated with the insects (Reymond, 2013; Felton et al., 2014).

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When phloem-feeding insects feed on plants, their stylets transiently puncture the

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epidermis and penetrate plant cell walls. The insects then ingest the phloem sap. During

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this process, insects secrete both gelling and watery saliva from their salivary glands

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into plant cells. The secreted gelling saliva quickly solidifies and forms a continuous

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salivary sheath in the plant encasing the full length of the stylet. The salivary sheath

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provides mechanical stability, and protection for the insect against plant chemical

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defenses. For example, inhibiting the expression of structural sheath protein (SHP), a

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salivary protein secreted by Acyrthosiphon pisum aphids, reduces their reproduction by

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disrupting salivary sheath formation and hence their feeding from host sieve tubes

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(Will and Vilcinskas, 2015). Watery saliva contains digestive, and cell wall-degrading

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enzymes. Plant immune responses to insect attack may be elicited or suppressed by

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compounds in insect saliva (Miles, 1999; Felton et al., 2014). Broadly speaking,

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effectors are proteins or other molecules produced by pathogens or insects that can alter

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host structures and functions (Hogenhout et al., 2009). Several insect effectors with

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diverse effects have been identified in aphids in recent years (Bos et al., 2010; Atamian

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et al., 2013; Rodriguez et al., 2014; Naessens et al., 2015). For example, the

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expression of aphid protein effector C002 in host plants increases the fecundity of green 4


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peach aphid, while another effector, Mp10, reduces aphid fecundity (Bos et al., 2010).

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Moreover, transient in planta expression of Mp10 activates jasmonic acid (JA) and

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salicylic acid (SA) signaling pathways (Rodriguez et al., 2014) and triggers chlorosis

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in Nicotiana benthamiana (Bos et al., 2010). Similarly, the expression of two candidate

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effectors, Me10 and Me23, from the potato aphid in host N. benthamiana plants

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increases aphid fecundity (Atamian et al., 2013), and MpMIF (a MIF cytokine secreted

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in aphid watery saliva during feeding) plays an important role in aphid survival and can

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affect both the SA and JA signaling pathways (Naessens et al., 2015). However, little is

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known about effectors from piercing-sucking herbivores other than aphids and their

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functions in host plants.

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Plants have evolved sophisticated defense mechanisms to protect themselves from

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insect herbivores, most of which are initiated by the recognition of their saliva or oral

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secretions. The signals are transmitted within plants via transduction networks

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including JA, ethylene (ET), SA, and hypersensitive response (HR) pathways.

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Accordingly, infestation by piercing-sucking insects increases the production of JA, SA,

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and ET in rice (Yuan et al., 2005; Du B et al., 2009; Hu et al., 2011). Key elements in

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these signaling pathways include mitogen-activated protein kinase (MAPK) cascades,

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which occur in all eukaryotes, are highly conserved, and modulate numerous cellular

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responses to diverse cues (Wu et al., 2007). These responses include complex defense

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responses against insects (Wu and Baldwin, 2010). For example, oral secretions from

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the chewing insect tobacco hornworm (Manduca sexta) induce MAPK-activated

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defense responses to herbivore attack in N. attenuata leaves (Wu et al., 2007).

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Similarly, aphid resistance conferred by the Mi-1 gene in tomato (Solanum

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lycopersicum) can be attenuated by virus-induced gene silencing (VIGS) of certain

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MAPKs and MAPK kinases (Li et al., 2006). MAPK cascades also play important

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roles in planthopper resistance gene-mediated immunity (Yuan et al., 2005).

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Mechanisms for resistance to phloem-feeding insects include the induction of forisome

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(sieve tube protein) dispersion, callose deposition, and thus, phloem plugging, which 5


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prevent insects from continuously ingesting phloem sap from plants (Will et al., 2007;

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Hao et al., 2008).

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The brown planthopper (BPH), Nilaparvata lugens (Stål), is a severe herbivorous

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insect pest of rice that causes extensive yield losses and economic damage to rice both

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directly (by feeding) and indirectly (by transmitting viral diseases). During outbreaks,

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planthoppers can completely destroy crops, an effect called “hopper burn” (Backus et

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al., 2005). Like other piercing-sucking insects, BPHs secrete gelling and watery saliva.

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Recently, genomic tools such as proteomics and transcriptomics have been used to

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investigate BPH salivary glands and saliva at the molecular level (Konishi et al., 2009;

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Ji et al., 2013; Huang et al., 2016; Liu et al., 2016). Two secretary proteins that

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actively participate in salivary sheath formation were recently identified in BPHs

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(Huang et al., 2015; Huang et al., 2016). Furthermore, several salivary proteins that

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play important roles in interactions between BPH and rice was identified (Petrova and

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Smith, 2014; Ji et al., 2017; Ye et al., 2017). However, the functions of the majority of

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BPH-secreted proteins have not yet been experimentally determined. The biological

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roles of specific BPH salivary protein effectors in rice-BPH interactions remain poorly

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understood.

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In an analysis of the BPH salivary gland transcriptome, we found a mucin-like

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protein gene highly expressed in BPH salivary glands. Mucins are a family of high

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molecular weight, heavily glycosylated proteins that mostly comprise tandem repeats

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of identical or highly similar sequences rich in serine, threonine, and proline residues

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(Verma and Davidson, 1994). Mucin-like proteins are widely distributed in

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eukaryotes, bacteria, and viruses. Intestinal mucins (IIM) and salivary gland mucins

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have been identified in insects. IIM is a major protein constituent of the peritrophic

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membrane that facilitates the digestive process, as well as protecting invertebrate

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digestive tracts from microbial infection (Wang and Granados, 1997). A mucin-like

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protein that was identified in the salivary glands of Anopheles gambiae through

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transcriptomic analysis might modulate parasite infectivity or help lubricate insect 6


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mouthparts (Francischetti et al., 2002). A mucin-like protein in the salivary proteome

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of BPH has been detected (Huang et al., 2016). However, the functions of mucin-like

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proteins in insects are largely unknown.

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Here, we identified this N. lugens-secreted mucin-like protein (abbreviated NlMLP)

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as an insect cell death-inducing protein involved in plant-insect interactions. NlMLP

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is required for salivary sheath formation and feeding of BPHs on their host plants.

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NlMLP induces defense responses in plant cells, including cell death, the expression of

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pathogen-responsive genes, and callose deposition. Finally, we found that the active

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part of NlMLP is located at its C-terminal region.

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RESULTS

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NlMLP is highly expressed in N. lugens salivary glands and secreted into rice

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tissues

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Sequencing of a cDNA library produced from BPH salivary glands yielded 40,000,000

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reads. After a series of assembly and alignment steps (Methods S1), 13,969 unigenes

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were functionally annotated with gene descriptions. Assignment of clusters of

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orthologous groups (COG) and gene ontology (GO) terms showed that the salivary

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gland proteins are involved in basic processes such as transcription and translation, as

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well functions including binding, catalytic activity, and secretion (Supplemental Fig.

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S1 and S2).

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Salivary proteins that are secreted outside of salivary gland cells to perform their

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functions should contain a secretory signal peptide; 399 unigenes in the BPH salivary

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gland transcriptome were predicted to encode proteins with signal peptides. Proteins

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with more than one predicted transmembrane domain, which are likely anchored in cell

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membranes of the salivary gland, were excluded. After these filtering steps, 256

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potential secretory proteins were retained (Table S1). A gene (CL865) showing high

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identity to the Laodelphax striatellus mucin-like protein was the most abundant in the

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transcriptome.

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We obtained a full-length cDNA for this gene, which contains a 2187 bp open

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reading frame (ORF) and encodes a polypeptide of 728 amino acid residues (Fig. 1A).

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We named this gene NlMLP (Nilaparvata lugens mucin-like protein) (accession

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number AK348750). The first 19 amino acids comprise the signal peptide, with

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cleavage predicted between residues 19 and 20. NlMLP is rich in serine (22.4%)

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residues, 36% of which are predicted to be potential mucin type O-glycosylation sites.

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Some repeated amino acid sequences, a typical feature of mucin-like proteins (Verma

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& Davidson, 1994), were found. NlMLP protein has been detected in both gelling and

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watery saliva (Huang et al., 2016; Liu et al., 2016). To investigate the functions of

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NlMLP, we analyzed mRNA levels in BPHs at various developmental stages including 8


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eggs, 1st to 5th instar BPHs, and female and male adults via quantitative

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reverse-transcription PCR (qRT-PCR). NlMLP expression was higher in insects at

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feeding stages (nymph or adult) than at the non-feeding stage (egg) (Fig. 1B). NlMLP

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transcripts were detected at higher levels in the salivary gland than in the gut, fat body,

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and remaining carcass (Fig. 1C). We also analyzed the expression of NlMLP in

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salivary glands by mRNA in situ hybridization. Hybridization signals were detected in

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the A-follicles of principal glands but not in the salivary ducts or accessory glands

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(Fig. 1D).

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To confirm that NlMLP was secreted into rice tissue during feeding, we extracted

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proteins from the leaf sheaths of plants following BPH feeding and analyzed them by

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mass spectrometry. Four NlMLP peptides were detected in BPH-infested rice leaf

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sheaths but not in non-infested rice (Fig. 1A), indicating that NlMLP was secreted into

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the rice plants.

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To determine the cellular localization of NlMLP in plant cells when transiently

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expressed, we conducted localization experiments using rice protoplasts and N.

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benthamiana leaf cells. When the NlMLP-GFP fusion protein was transiently

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expressed in rice protoplasts, GFP fluorescence was detected only in the cytoplasm,

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while control GFP fluorescence was detected in both the cytoplasm and nucleus (Fig.

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1E). When the NlMLP-YFP fusion protein was transiently expressed in N.

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benthamiana leaves via agroinfiltration, NlMLP localized to the cytoplasm

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(Supplemental Fig. S3).

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NlMLP is required for the feeding of BPHs on rice plants and for insect

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performance

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To elucidate the role of NlMLP in BPH, we synthesized double-stranded RNA (dsRNA)

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from NlMLP and injected it into 4th instar BPH nymphs to mediate RNA interference

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(RNAi). This treatment had a very strong silencing effect, reducing NlMLP transcript

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levels significantly (~95%) on the first day after treatment compared to the levels in 9


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two control groups receiving either no injection or injection with dsGFP (P < 0.001 for

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C and dsMLP from 1 to 6 days; P = 0.017 for dsGFP and dsMLP at 1 and 5 days; P =

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0.016 for dsGFP and dsMLP at 2, 3 and 6 days; P = 0.015 for dsGFP and dsMLP at 4

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days; Supplemental Fig. S4). The silencing was confirmed by RNA gel blot analysis

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two days after injection (Supplemental Fig. S4). The treated BPH insects were allowed

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to feed on TN1 rice plants. The survival rate of BPHs harboring a silenced NlMLP gene

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was significantly lower (from 2 to 10 days following injection) than those of the two

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control groups (P < 0.001 for C and dsMLP and P = 0.046 for dsGFP and dsMLP at 2

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days; P < 0.001 for C and dsMLP and P = 0.001 for dsGFP and dsMLP at 10 days;

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Fig. 2A). The cumulative mortality rate of BPHs injected with dsMLP, dsGFP, and

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non-injected BPHs was 96%, 60%, and 52%, respectively, at 10 days after injection

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(Fig. 2A). BPHs subjected to the NlMLP RNAi treatment also excreted significantly

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less honeydew, a simple measurable indicator of BPH feeding activity, than the two

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control groups (P < 0.001 for C and dsMLP; P = 0.003 for dsGFP and dsMLP; Fig.

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2B), as well as having smaller weight gain values (P = 0.011 for C and dsMLP; P =

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0.031 for dsGFP and dsMLP; Fig. 2C) and weight gain ratios (P = 0.023 for C and

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dsMLP; P = 0.043 for dsGFP and dsMLP; Fig. 2D). Furthermore, silencing of NlMLP

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reduced BPH virulence. Rice plants died in 7 days after infested by common BPH

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insects or BPH insects injected with dsGFP, while those plants infested by BPH

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insects injected with dsMLP still survived and grew normally (Supplemental Fig. S5).

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These results indicate that silencing the NlMLP gene significantly reduced the feeding

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and performance of BPHs on rice plants.

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Feeding on rice plants expressing dsRNAs was previously shown to trigger RNA

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interference of a target gene in BPH (Zha et al., 2011). We therefore transformed

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BPH-susceptible rice plants with NlMLP-dsRNA and selected a T2 homozygous

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dsMLP-transgenic

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(Supplemental Fig. S6A). When 2nd instar BPHs were fed on dsMLP-transgenic plants,

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their expression level of NlMLP was significantly (40%) lower than in BPHs fed on

line

expressing

NlMLP-dsRNA

via

qRT-PCR

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analysis

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wild-type plants at 7 and 9 days after the start of exposure (P = 0.042 at 7 days; P =

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0.045 at 9 days; Supplemental Fig. S6B). BPH survival and weight gain were also

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significantly lower in insects fed on dsMLP-transgenic plants than in those fed on

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wild-type plants from 7 to 10 days (P = 0.049 at 7 days; P = 0.046 at 8 days; P = 0.005

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at 9 days; P = 0.008 at 10 days; Fig. 2E) and after 10 days (P = 0.023; Fig. 2F),

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respectively. These results clearly show that NlMLP protein is essential for BPH

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feeding and performance.

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NlMLP is necessary for salivary sheath formation

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NlMLP was found in both gelling saliva and watery saliva (Huang et al., 2016). To

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further investigate the effects of NlMLP on feeding, we focused on salivary sheath

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formation. First, we fed BPHs on an artificial diet in Parafilm sachets for 2 days and

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analyzed their salivary sheaths by fluorescence microscopy and scanning electron

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microscopy observation. The fluorescence microscopy analysis revealed that BPHs

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subjected to the NlMLP RNAi treatment produced salivary sheaths that were

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significantly shorter and less branched than those produced by the control BPHs

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receiving either no injection or injection with dsGFP (Fig. 3, A and B). Moreover, the

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structure of the sheaths was incomplete or predominantly amorphous, or gelling saliva

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deposits at their stylet penetration sites were minimal, whereas those secreted by

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control BPHs had complete, typical structures (Fig. 3, C-E). Second, we collected

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stems from rice plants after BPH feeding and sectioned them to observe salivary sheath

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morphology in planta. Most salivary sheaths in rice stems produced by the control

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BPHs reached the phloem (Fig. 3, F and G), whereas most salivary sheaths produced

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by NlMLP-silenced BPHs were shorter and failed to reach the phloem, instead stopping

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in the rice epidermis or xylem (Fig. 3H). Together, these observations indicate that

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NlMLP is necessary for salivary sheath formation. Silencing of NlMLP in BPHs

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resulted in imperfect, short salivary sheaths, thus affecting phloem feeding.

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NlMLP induces plant cell death

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The above findings clearly show that NlMLP is secreted into rice tissues (Fig. 1A) and

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that it is localized to the cytoplasm of rice cells (Fig. 1E). To uncover the potential role

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of NlMLP in the host plant, we transiently expressed NlMLP without the signal peptide

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in rice protoplasts. We measured cell death, fluorescein diacetate (FDA) staining of the

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protoplasts showed that the cell viability of protoplasts expressing NlMLP was

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significantly lower than that of control protoplasts expressing GFP (P < 0.001; Fig. 4,

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A and B). We also co-expressed NlMLP together with the luciferase (LUC) gene in rice

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protoplasts. LUC activity was significantly lower in protoplasts co-expressing NlMLP

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compared to the control co-expressing GFP (P < 0.001; Fig. 4C). Immunoblotting

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confirmed that NlMLP and GFP were expressed properly in the rice protoplasts

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(Supplemental Fig. S8A). These observations indicate that NlMLP expression triggers

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cell death in rice protoplasts. To determine whether NlMLP-triggered cell death is

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affected by the presence of BPH-resistance genes in rice, we performed similar LUC

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assays after co-transfection of protoplasts with NlMLP and the genes Bph6, Bph9, and

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Bph14. The LUC activity was still significantly lower in the presence of NlMLP than in

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the GFP controls, regardless of the presence of resistance genes (P = 0.001 for Bph6; P

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= 0.005 for Bph9; P = 0.004 for Bph14; Supplemental Fig. S7). Therefore, NlMLP

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expression triggers cell death in rice protoplasts independently of these BPH-resistance

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genes.

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We further verified the ability of NlMLP to induce plant cell death by performing A.

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tumefaciens-mediated expression of NlMLP in N. benthamiana leaves. INF1, an elicitin

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secreted by Phytophthora infestans that induces HR cell death in Nicotiana plants

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(Derevnina et al., 2016), was used as a positive control, while GFP was used as a

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negative control. NlMLP, with or without YFP-HA tag, triggered marked cell death

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(Fig. 4, D and E). Moreover, ion leakage was significantly higher from leaves

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expressing NlMLP or INF1 than from the GFP-expressing controls (P = 0.002 for GFP

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and INF1; P < 0.001 for GFP and NlMLP; Fig. 4F). Immunoblot analysis showed that 12


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GFP, INF1, and NlMLP proteins accumulated to comparable degrees in N.

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benthamiana leaves (Supplemental Fig. S8B). However, the cell death symptoms

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caused by INF1 and NlMLP is different. Leaves infiltrated with INF1 strain appeared

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chlorosis in 4 days, and became severe necrosis accompanied by brown or black color

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after 5 days. On leaves infiltrated with NlMLP, white or grey-white necrotic spots

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appeared in 4 days and became bigger around the infiltrated site as time goes on

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(Supplemental Fig. S9). We further investigated the quantity of NlMLP required for

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the cell death symptoms. We set up different concentrations (OD600=0.005, 0.01, 0.02,

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0.03, 0.04, 0.05, 0.08, 0.1, 0.2 and 0.3) of NlMLP strains to infect N. benthamiana

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leaves, and found that cell death was caused by NlMLP strains in OD=0.01 or over

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but was not when the OD is 0.005 (Supplemental Fig. S10A). Immunoblot detected

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NlMLP protein in the leaves infected by in leaves infected with strains in OD 0.01 or

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over, but not in 0.005 (Supplemental Fig. S10B).

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To further characterize the physiological properties of the cell death induced by

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NlMLP, we examined the effects of treatments that inhibit various potential cell

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death-associated processes in rice protoplasts and N. benthamiana leaves. The

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application of LaCl3 blocked the induction of cell death by NlMLP, suggesting that the

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cell-death process mediated by NlMLP is dependent on a calcium signaling pathway

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(Boudsocq et al., 2010) (Table 1). There was no difference in cell viability between

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protoplasts incubated in the light or dark, indicating that the cell death process induced

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by NlMLP is light-independent (Asai et al., 2000). Bcl-xl is an anti-apoptotic protein

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(Chen et al., 2012). The expression of Bcl-xl in N. benthamiana leaves suppressed cell

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death induced by subsequent NlMLP expression (Table 1). Our results indicate that cell

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death induced by NlMLP shares some common properties with cell death induced by

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BAX and INF1. MAPK cascades play important roles in defense-related signal

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transduction (Yang et al., 2001). MEK2 is a MAPK kinase that acts upstream of

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SA-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) and

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controls multiple defense responses to pathogen invasion (Yang et al., 2001). When we 13


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silenced MEK2 in N. benthamiana plants via VIGS (P = 0.006; Fig. 4I),

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NlMLP-triggered cell death was significantly reduced in MEK2-silenced plants (Fig.

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4H), but not in control plants (Fig. 4G). However, the presence of INF1, which triggers

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cell death independently of MEK2 (Takahashi et al., 2007), clearly caused necrosis in

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MEK2-silenced plants (Fig. 4H). Therefore, NlMLP-triggered cell death is associated

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with MEK2-dependent MAPK cascades.

349 350

NlMLP triggers plant defense responses

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Callose deposition is used as a marker for plant basal defense responses and participates

352

in plant defenses against phloem sap ingestion by insects (Hann and Rathjen, 2007;

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Hao et al., 2008). Thus, to determine whether NlMLP activates defense responses in

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planta, we expressed NlMLP in N. benthamiana leaves and investigated callose

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deposition by aniline blue staining. Many more callose spots were present in

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NlMLP-expressing leaves (47.0 per infiltration) than in GFP-expressing leaves (3.5 per

357

infiltration) (P = 0.001; Fig. 5A). Moreover, NlMLP induced transcriptional activation

358

of the pathogen resistance (PR) genes NbPR3 (P = 0.143 at 24h; P = 0.002 at 48h) and

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NbPR4 (P = 0.002 at 24h; P = 0.002 at 48h), but not NbPR1 (P = 0.625 at 24h; P =

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0.180 at 48h), within 48 h of infection (Fig. 5B). The upregulation of genes encoding

361

acidic NbPR1 protein is a characteristic feature of the activated SA-signaling pathway,

362

while the induction of genes encoding basic NbPR3 and NbPR4 proteins is associated

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with JA-dependent defense responses (Zhang et al., 2012; Naessens et al., 2015). Thus,

364

NlMLP appears to induce defense responses mediated by the JA signaling pathway,

365

thereby promoting the production of PR proteins and the biosynthesis of cell

366

wall-reinforcing callose.

367 368

The functional motif is located in the C-terminus of NlMLP

369

NlMLP showed no sequence similarity to any known cell death-inducing effector. To

370

delineate the functional domains of NlMLP, we assayed the ability of N-terminal and 14


371

C-terminal deletion mutant proteins to trigger cell death in N. benthamiana leaves (Fig.

372

6). The N-terminal deletion mutant M428-728 strongly triggered cell death, but the

373

C-terminal deletion mutants M32-319 and M319-428 did not. Moreover, M428-674 triggered

374

cell death less strongly than did M428-728, and the M674-728 mutant did not induce cell

375

death at all. These findings suggest that the 428-674 amino-acid fragment is required

376

for triggering cell death and that the 674-728 amino-acid fragment might promote this

377

effect. We also found that the 428-674 fragment was required for the expression of

378

NbPR4 (P = 0.001 for GFP and M1-728; P = 0.002 for GFP and M32-728; P = 0.008 for

379

GFP and M319-728; P < 0.001 for GFP and M428-728; P = 0.129 for GFP and M674-728; P

380

= 0.007 for GFP and M428-674; P = 0.209 for GFP and M319-428; P = 0.656 for GFP and

381

M32-319; Fig. 6). Immunoblot analysis showed that the mutant proteins accumulated to

382

comparable levels in N. benthamiana leaves (Supplemental Fig. S8C).

15


383

DISCUSSION

384

This is the first report of a planthopper salivary protein that plays important roles in

385

feeding and interactions with the host plant. We identified a N. lugens mucin-like

386

protein, NlMLP, which is highly expressed in the salivary glands of BPHs and secreted

387

into rice tissue during BPH feeding. NlMLP is necessary for the probing of rice plants

388

by BPHs to obtain phloem sap for insect survival. BPH feeding was inhibited and insect

389

performance was significantly reduced when NlMLP expression was knocked-down

390

(Fig. 2). Furthermore, the salivary sheaths produced by NlMLP-silenced BPHs were

391

shorter and less branched than those of control BPHs fed on both an artificial diet and

392

rice tissue. The sheaths had incomplete structures and were predominantly

393

amorphous.

394

Mucin-like proteins have been identified in various organisms; some mucins are

395

involved in controlling mineralization (Boskey, 2003) and are associated with

396

processes including nacre formation in mollusks (Marin et al., 2000), calcification in

397

echinoderms (Boskey, 2003), and bone formation in vertebrates (Midura and Hascall,

398

1996). Based on our current results, mucin appears to be an essential component of

399

the BPH salivary sheath. Notably, the mucin-like protein NlMLP is rich in serine,

400

which provides attachment sites for carbohydrate chains that participate in the

401

formation of large extracellular aggregates (Korayem et al., 2004), thus functioning in

402

the formation of salivary sheaths, which support stylet movements and exploration of

403

the host plant tissue. Therefore, reductions in the levels of NlMLP may prevent the

404

construction of complete sheaths. The immediate consequence of this reduction in

405

NlMLP-RNAi insects is that salivary sheaths formed in rice plants do not reach into the

406

sieve tube, which likely accounts for the reduction in phloem feeding and performance

407

of these insects on rice plants.

408

In aphids, the salivary protein SHP contributes to the solidification of gelling saliva

409

and sheath formation, partly through the formation of disulfide cystine bonds (Carolan

410

et al., 2009; Will and Vilcinskas, 2015). Mucins can form disulfide-dependent soluble 16


411

dimers and multimeric insoluble gels through cross-linking of cysteines (Axelsson et

412

al., 1998). Cysteine residues in NlMLP might behave in a similar fashion and strongly

413

contribute to the formation of the polymeric matrix during sheath hardening via the

414

formation of intermolecular disulfide bonds. NlMLP might also function in the growth

415

and development of BPHs. Silencing of NlMLP affected insect development, which in

416

turn reduced salivary sheath formation, feeding, and performance.

417

Plants usually detect molecules emitted by parasites to trigger defense responses.

418

When BPHs feed on phloem sap, their saliva is secreted into rice tissue, which contains

419

bioactive components involved in inducing the expression of defense genes. We

420

demonstrated that NlMLP is one such bioactive component. Mucin-like proteins have

421

also been detected in fungal pathogens. The surfaces of many parasites, including the

422

protozoan parasite Trypanosoma cruzi (Buscaglia et al., 2006), the fish pathogen T.

423

carassii (Lischke et al., 2000), and the potato pathogen Phytophthora infestans

424

(Gornhardt et al., 2000) are covered in mucins, which participate in interactions with

425

host cells during the invasion process (Buscaglia et al., 2006; Larousse et al., 2014).

426

Similarly, NlMLP is highly expressed in salivary glands and is secreted into the plant.

427

We found that NlMLP expression triggered cell death in rice protoplasts and N.

428

benthamiana leaves (Fig. 4), as well as plant immunity responses, including the

429

induction of PR gene expression and callose synthesis in leaves (Fig. 5). NlMLP

430

molecules on the surface of the salivary sheath, representing an essential component

431

of this structure, can be detected as signal of BPH feeding by host cells and evoke

432

defense responses. NlMLP in watery saliva may play the same role as well. Our

433

results indicate that the functional portion of NlMLP is located at its C-terminus. The

434

presence of a 428-674 amino-acid fragment was sufficient for triggering cell death and

435

inducing the expression of NbPR4. Mucin-like proteins are also detected in other

436

piercing-sucking insects such as leafhopper (Hattori et al., 2015) and several mosquito

437

species (Das et al., 2010). The wide taxonomic range of hosts in which NlMLP can

438

trigger cell death suggests that NlMLP may be recognized by a conserved protein found 17


439

in many plants. The plant receptor that recognizes NlMLP remains to be identified.

440

The defense reaction elicited by NlMLP shares common features with immune

441

responses which shown by well-known effectors and pathogen-associated molecular

442

patterns (PAMPs). NlMLP might be an elicitor that involved in the PTI process. It

443

may be recognized by plant PRRs which triggers plant defensive responses. NlMLP

444

triggers cell death, a common phenomenon in effector-triggered immune responses.

445

Ca2+ is a well-known secondary signal in eukaryotes. The early defense response to

446

BPH in rice involves Ca2+ influx, which is a common early plant response triggered

447

by insect feeding (Hao et al., 2008; Hogenhout and Bos, 2011; Bonaventure, 2012).

448

Our results indicate that the cell death process induced by NlMLP is dependent on

449

Ca2+ influx. Moreover, cell death induced by NlMLP is not dependent on light and is

450

suppressed by the anti-apoptotic protein Bcl-xl. NlMLP-triggered cell death requires

451

the MEK2 MAP kinase signal transduction pathway. MEK2 is a common component in

452

MAP kinase pathways required for HR followed by certain Avr-R interactions in

453

tomato and tobacco (Morris, 2001; Del Pozo et al., 2004; King et al., 2014). NbMEK2

454

is also required for cell death triggered by the Phytophthora sojae RxLR effector

455

Avh241 in N. benthamiana (Yu et al., 2012). Given the broad range of plants

456

responding to NlMLP and the dependence of these responses on the MEK2 pathway,

457

we speculate that plants respond to NlMLP may via a conserved upstream component

458

of plant signaling pathways. The expression of NlMLP induced the JA pathway marker

459

genes NbPR3 and NbPR4. NbPR3 is a chitinase gene associated with JA-dependent

460

defenses that is induced by elicitors (Naessens et al., 2015), while NbPR4 encodes a

461

hevein-like chitinase that is also primarily associated with JA responses (Kiba et al.,

462

2014). The findings suggest that defense responses triggered by NlMLP are associated

463

with the JA signaling pathway. NlMLP expression in N. benthamiana leaves also

464

induced callose deposition. Callose deposition is a useful mechanism conferring plant

465

immunity to insects and pathogens (Luna et al., 2011). In the interaction of rice and

466

BPH, callose deposition on the sieve plates occludes sieve tubes, thereby directly 18


467

inhibiting continuous feeding by BPHs (Hao et al., 2008).

468

Nevertheless, BPH can successfully attack most rice cultivars and even results in

469

hopper burn. This success is thought because of the evolved ability of BPH to suppress

470

or counteract rice defense responses triggered by NlMLP and other elicitors (Walling,

471

2008 Kaloshian and Walling, 2016). Hemipterans saliva is a complex mixture of

472

biomolecules with potential roles in overcoming plant immune responses and enables

473

hemipterans to manipulate host responses to their advantage (Miles 1999; Kaloshian

474

and Walling, 2016). One example is that BPH can decompose the deposited callose and

475

unplug sieve tube occlusions by activating b-1,3-glucanase genes and thereby facilitate

476

the continuous phloem-feeding in rice plants (Hao et al., 2008). To date, some effectors,

477

including C002, Me10, Me23, Mp1, Mp2, and Mp55, have been identified by assays in

478

planta overexpression and RNAi in aphids. These effectors contribute to aphid survival

479

and suppressing host defense (Mutti et al., 2008; Bos et al., 2010; Atamian et al., 2013;

480

Pitino and Hogenhout, 2013; Elzinga et al., 2014). The effectors may target any

481

component in pattern-triggered immunity or host cell trafficking pathway (Derevnin et

482

al., 2016; Kaloshian and Walling, 2016; Rodriguez et al., 2017). It is likely that potent

483

effectors in BPH also target such cell processes in rice and enable BPH to successfully

484

feed on rice plants, which induces cascade reactions termed the BPH-feeding cascade,

485

and results in the death of susceptible rice varieties (Cheng et al., 2013). In contrast, in

486

resistant rice the resistance gene induces a strong defense response that inhibits insect

487

feeding, growth, and development, and enables the plant to grow normally.

488

In summary, our results indicate that NlMLP secreted by BPHs into rice plants plays

489

dual roles as a component in feeding sheath formation and activating plant defense

490

responses. The defense responses induced by NlMLP in plant cells are related to Ca2+

491

mobilization and the MEK2 MAP kinase and JA signaling pathways. Further studies

492

are needed to identify the target of NlMLP in rice cells and BPH effectors that suppress

493

rice defense responses to further explore the interaction mechanisms between plants

494

and planthoppers. 19


495

MATERIALS AND METHODS

496

Plant materials and insects

497

Wild-type and transgenic rice (Oryza sativa) plants were grown in the experimental

498

fields at Wuhan University Institute of Genetics, China under routine management

499

practices. Brown planthopper biotype 1 insect populations were reared on

500

one-month-old plants of the susceptible rice variety TN1 under controlled

501

environmental conditions (26 ± 0.5°C, 16 h light/8 h dark cycle).

502 503

Tanscriptome analysis of BPH salivary glands

504

BPH adult females were dipped in 70% ethanol for several seconds and washed in

505

0.85% NaCl solution. Salivary glands were dissected in phosphate buffer saline

506

solution (pH 7.4). This was accomplished by pulling the head off of the thorax with

507

forceps and carefully removing the salivary glands that emerged from the distal region

508

of the severed head. A total of 2,000 salivary glands were dissected and directly dipped

509

into 300 µL RNAiso Plus (Takara) for RNA preparation. The poly (A) RNA was

510

isolated, broken into smaller fragments (200-700 bp), and reverse-transcribed to

511

synthesize cDNA for Illumina HiSeq™ 2000 sequencing. After stringent quality

512

assessment and data filtering, 40,000,000 reads were selected as high-quality reads

513

(after removing adapters and low-quality regions) for further analysis. A set of

514

12,668,039 high-quality reads with an average length of 400 bp was assembled,

515

resulting in 59,510 contigs. The contigs were assembled into 31,645 unigenes,

516

consisting of 3960 clusters and 27,685 singletons. All unigene sequences were aligned

517

by Blastx to protein sequences in databases including nr, Swiss-Prot, KEGG, and COG,

518

followed by sequences in the non-redundant NCBI protein database (with e