A mucin-like protein of planthopper is required for ... · insect pest of rice that causes...
Transcript of A mucin-like protein of planthopper is required for ... · insect pest of rice that causes...
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Short title: Planthopper MLP induces plant immunity response 1
Correspondence: Guangcun He, National Key Laboratory of Hybrid Rice, College of 2
Life Sciences, Wuhan University, Wuhan 430072, China 3
E-mail: [email protected] 4
Tel: +86-27-68752384 5
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A mucin-like protein of planthopper is required for feeding and induces immunity 7
response in plants 8
Xinxin Shangguan1, Jing Zhang
1, Bingfang Liu
1, Yan Zhao
1, Huiying Wang
1, 9
Zhizheng Wang1, Jianping Guo
1, Weiwei Rao
1, Shengli Jing
1, Wei Guan
1, Yinhua Ma
1, 10
Yan Wu1, Liang Hu
1, Rongzhi Chen
1, Bo Du
1, Lili Zhu
1, Dazhao Yu
2 & Guangcun 11
He1*
12
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1State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, 14
430072 Wuhan, China; 2Institute for Plant Protection and Soil Sciences, Hubei 15
Academy of Agricultural Sciences, 430064 Wuhan, China. 16
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One sentence Summary: A secreted mucin-like protein in the rice brown planthopper 18
(Nilaparvata lugens) enables insect feeding and induces plant immune responses. 19
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Footnotes: 21
Author contributions: G.H. conceived the original research plans and supervised the 22
experiments. G.H. and X.S. designed the experiments. X.S. carried out most of the 23
experiments. J.Z., B.L., H.W., Z.W., S.J., W.G., R.C., B.D., L.Z., and D.Y. carried out 24
some of the experiments. G.H. and X.S. analyzed data and wrote the manuscript. 25
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Funding information: This work was supported by grants from National Program on 27
Research & Development of Transgenic Plants Grants (2016ZX08009003-001-008), 28
National Natural Science Foundation of China (31630063, 31230060, 31401732), and 29
National Key Research and Development Program (2016YFD0100600, 30
2016YFD0100900). 31
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Corresponding author: Guangcun He ([email protected]) 33
Plant Physiology Preview. Published on November 13, 2017, as DOI:10.1104/pp.17.00755
Copyright 2017 by the American Society of Plant Biologists
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Glossary 34
BPH: brown planthopper; 35
ET: ethylene; 36
GO: gene ontology; 37
COG: clusters of orthologous groups; 38
dsRNA: double-stranded RNA; 39
FDA: fluorescein diacetate; 40
GFP: green fluorescent protein; 41
HR: hypersensitive response; 42
IIM: Intestinal mucins; 43
LUC: luciferase; 44
JA: jasmonic acid; 45
MAPK: mitogen-activated protein kinase; 46
NlMLP: N. lugens-secreted mucin-like protein; 47
ORF: open reading frame; 48
PAMPs: pathogen-associated molecular patterns; 49
PRR: Pattern recognition receptor 50
PTI: PAMP-triggered immunity 51
RLUC: Renilla luciferase gene 52
RNAi: RNA interference; 53
qRT-PCR: quantitative reverse-transcription PCR; 54
SA: salicylic acid; 55
SEM: scanning electron microscopy; 56
SHP: structural sheath protein; 57
SIPK: SA-induced protein kinase; 58
Ubi: ubiquitin; 59
VIGS: virus-induced gene silencing; 60
WIPK: wounding-induced protein kinase 61
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Abstract 65
The brown planthopper, Nilaparvata lugens (Stål), is a pest that threatens rice 66
production worldwide. While feeding on rice plants, planthoppers secrete saliva, which 67
plays crucial roles in nutrient ingestion and modulating plant defense responses, 68
although the specific functions of salivary proteins remain largely unknown. We 69
identified a N. lugens-secreted mucin-like protein (NlMLP) by transcriptome and 70
proteome analyses and characterized its function, both in brown planthopper and in 71
plants. NlMLP is highly expressed in salivary glands and is secreted into rice during 72
feeding. Inhibition of NlMLP expression in planthoppers disturbs the formation of 73
salivary sheaths, thereby reducing their performance. In plants, NlMLP induces cell 74
death, the expression of defense-related genes, and callose deposition. These defense 75
responses are related to Ca2+
mobilization and the MEK2 MAP kinase and JA 76
signaling pathways. The active region of NlMLP that elicits plant responses is located 77
in its C-terminus. Our work provides a detailed characterization of a salivary protein 78
from a piercing-sucking insect other than aphids. Our findings that the protein that 79
functions in plant immune responses offer new insights into the mechanism 80
underlying interactions between plants and herbivorous insects. 81
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Introduction 82
Plants are subjected to attack by diverse herbivorous insects, which are generally 83
classified based on their feeding strategies as chewing or piercing-sucking insects. 84
Chewing insects, such as caterpillars and beetles, can cause serious mechanical damage 85
to plant tissues, whereas piercing-sucking insects feed on plants through specially 86
adapted mouthparts known as stylets and cause only limited physical damage to plant 87
tissues (Walling, 2000). Insects can also injure plants indirectly by transmitting viral, 88
bacterial, and fungal pathogens. Plants use sophisticated perception systems to detect 89
insect feeding through cues derived not only from damage caused by feeding 90
(Reymond et al., 2000), but also from insect saliva, oral secretions, eggs, volatiles, and 91
microbes associated with the insects (Reymond, 2013; Felton et al., 2014). 92
When phloem-feeding insects feed on plants, their stylets transiently puncture the 93
epidermis and penetrate plant cell walls. The insects then ingest the phloem sap. During 94
this process, insects secrete both gelling and watery saliva from their salivary glands 95
into plant cells. The secreted gelling saliva quickly solidifies and forms a continuous 96
salivary sheath in the plant encasing the full length of the stylet. The salivary sheath 97
provides mechanical stability, and protection for the insect against plant chemical 98
defenses. For example, inhibiting the expression of structural sheath protein (SHP), a 99
salivary protein secreted by Acyrthosiphon pisum aphids, reduces their reproduction by 100
disrupting salivary sheath formation and hence their feeding from host sieve tubes 101
(Will and Vilcinskas, 2015). Watery saliva contains digestive, and cell wall-degrading 102
enzymes. Plant immune responses to insect attack may be elicited or suppressed by 103
compounds in insect saliva (Miles, 1999; Felton et al., 2014). Broadly speaking, 104
effectors are proteins or other molecules produced by pathogens or insects that can alter 105
host structures and functions (Hogenhout et al., 2009). Several insect effectors with 106
diverse effects have been identified in aphids in recent years (Bos et al., 2010; Atamian 107
et al., 2013; Rodriguez et al., 2014; Naessens et al., 2015). For example, the 108
expression of aphid protein effector C002 in host plants increases the fecundity of green 109
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peach aphid, while another effector, Mp10, reduces aphid fecundity (Bos et al., 2010). 110
Moreover, transient in planta expression of Mp10 activates jasmonic acid (JA) and 111
salicylic acid (SA) signaling pathways (Rodriguez et al., 2014) and triggers chlorosis 112
in Nicotiana benthamiana (Bos et al., 2010). Similarly, the expression of two candidate 113
effectors, Me10 and Me23, from the potato aphid in host N. benthamiana plants 114
increases aphid fecundity (Atamian et al., 2013), and MpMIF (a MIF cytokine secreted 115
in aphid watery saliva during feeding) plays an important role in aphid survival and can 116
affect both the SA and JA signaling pathways (Naessens et al., 2015). However, little is 117
known about effectors from piercing-sucking herbivores other than aphids and their 118
functions in host plants. 119
Plants have evolved sophisticated defense mechanisms to protect themselves from 120
insect herbivores, most of which are initiated by the recognition of their saliva or oral 121
secretions. The signals are transmitted within plants via transduction networks 122
including JA, ethylene (ET), SA, and hypersensitive response (HR) pathways. 123
Accordingly, infestation by piercing-sucking insects increases the production of JA, SA, 124
and ET in rice (Yuan et al., 2005; Du B et al., 2009; Hu et al., 2011). Key elements in 125
these signaling pathways include mitogen-activated protein kinase (MAPK) cascades, 126
which occur in all eukaryotes, are highly conserved, and modulate numerous cellular 127
responses to diverse cues (Wu et al., 2007). These responses include complex defense 128
responses against insects (Wu and Baldwin, 2010). For example, oral secretions from 129
the chewing insect tobacco hornworm (Manduca sexta) induce MAPK-activated 130
defense responses to herbivore attack in N. attenuata leaves (Wu et al., 2007). 131
Similarly, aphid resistance conferred by the Mi-1 gene in tomato (Solanum 132
lycopersicum) can be attenuated by virus-induced gene silencing (VIGS) of certain 133
MAPKs and MAPK kinases (Li et al., 2006). MAPK cascades also play important 134
roles in planthopper resistance gene-mediated immunity (Yuan et al., 2005). 135
Mechanisms for resistance to phloem-feeding insects include the induction of forisome 136
(sieve tube protein) dispersion, callose deposition, and thus, phloem plugging, which 137
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prevent insects from continuously ingesting phloem sap from plants (Will et al., 2007; 138
Hao et al., 2008). 139
The brown planthopper (BPH), Nilaparvata lugens (Stål), is a severe herbivorous 140
insect pest of rice that causes extensive yield losses and economic damage to rice both 141
directly (by feeding) and indirectly (by transmitting viral diseases). During outbreaks, 142
planthoppers can completely destroy crops, an effect called “hopper burn” (Backus et 143
al., 2005). Like other piercing-sucking insects, BPHs secrete gelling and watery saliva. 144
Recently, genomic tools such as proteomics and transcriptomics have been used to 145
investigate BPH salivary glands and saliva at the molecular level (Konishi et al., 2009; 146
Ji et al., 2013; Huang et al., 2016; Liu et al., 2016). Two secretary proteins that 147
actively participate in salivary sheath formation were recently identified in BPHs 148
(Huang et al., 2015; Huang et al., 2016). Furthermore, several salivary proteins that 149
play important roles in interactions between BPH and rice was identified (Petrova and 150
Smith, 2014; Ji et al., 2017; Ye et al., 2017). However, the functions of the majority of 151
BPH-secreted proteins have not yet been experimentally determined. The biological 152
roles of specific BPH salivary protein effectors in rice-BPH interactions remain poorly 153
understood. 154
In an analysis of the BPH salivary gland transcriptome, we found a mucin-like 155
protein gene highly expressed in BPH salivary glands. Mucins are a family of high 156
molecular weight, heavily glycosylated proteins that mostly comprise tandem repeats 157
of identical or highly similar sequences rich in serine, threonine, and proline residues 158
(Verma and Davidson, 1994). Mucin-like proteins are widely distributed in 159
eukaryotes, bacteria, and viruses. Intestinal mucins (IIM) and salivary gland mucins 160
have been identified in insects. IIM is a major protein constituent of the peritrophic 161
membrane that facilitates the digestive process, as well as protecting invertebrate 162
digestive tracts from microbial infection (Wang and Granados, 1997). A mucin-like 163
protein that was identified in the salivary glands of Anopheles gambiae through 164
transcriptomic analysis might modulate parasite infectivity or help lubricate insect 165
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mouthparts (Francischetti et al., 2002). A mucin-like protein in the salivary proteome 166
of BPH has been detected (Huang et al., 2016). However, the functions of mucin-like 167
proteins in insects are largely unknown. 168
Here, we identified this N. lugens-secreted mucin-like protein (abbreviated NlMLP) 169
as an insect cell death-inducing protein involved in plant-insect interactions. NlMLP 170
is required for salivary sheath formation and feeding of BPHs on their host plants. 171
NlMLP induces defense responses in plant cells, including cell death, the expression of 172
pathogen-responsive genes, and callose deposition. Finally, we found that the active 173
part of NlMLP is located at its C-terminal region. 174
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RESULTS 175
NlMLP is highly expressed in N. lugens salivary glands and secreted into rice 176
tissues 177
Sequencing of a cDNA library produced from BPH salivary glands yielded 40,000,000 178
reads. After a series of assembly and alignment steps (Methods S1), 13,969 unigenes 179
were functionally annotated with gene descriptions. Assignment of clusters of 180
orthologous groups (COG) and gene ontology (GO) terms showed that the salivary 181
gland proteins are involved in basic processes such as transcription and translation, as 182
well functions including binding, catalytic activity, and secretion (Supplemental Fig. 183
S1 and S2). 184
Salivary proteins that are secreted outside of salivary gland cells to perform their 185
functions should contain a secretory signal peptide; 399 unigenes in the BPH salivary 186
gland transcriptome were predicted to encode proteins with signal peptides. Proteins 187
with more than one predicted transmembrane domain, which are likely anchored in cell 188
membranes of the salivary gland, were excluded. After these filtering steps, 256 189
potential secretory proteins were retained (Table S1). A gene (CL865) showing high 190
identity to the Laodelphax striatellus mucin-like protein was the most abundant in the 191
transcriptome. 192
We obtained a full-length cDNA for this gene, which contains a 2187 bp open 193
reading frame (ORF) and encodes a polypeptide of 728 amino acid residues (Fig. 1A). 194
We named this gene NlMLP (Nilaparvata lugens mucin-like protein) (accession 195
number AK348750). The first 19 amino acids comprise the signal peptide, with 196
cleavage predicted between residues 19 and 20. NlMLP is rich in serine (22.4%) 197
residues, 36% of which are predicted to be potential mucin type O-glycosylation sites. 198
Some repeated amino acid sequences, a typical feature of mucin-like proteins (Verma 199
& Davidson, 1994), were found. NlMLP protein has been detected in both gelling and 200
watery saliva (Huang et al., 2016; Liu et al., 2016). To investigate the functions of 201
NlMLP, we analyzed mRNA levels in BPHs at various developmental stages including 202
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eggs, 1st to 5th instar BPHs, and female and male adults via quantitative 203
reverse-transcription PCR (qRT-PCR). NlMLP expression was higher in insects at 204
feeding stages (nymph or adult) than at the non-feeding stage (egg) (Fig. 1B). NlMLP 205
transcripts were detected at higher levels in the salivary gland than in the gut, fat body, 206
and remaining carcass (Fig. 1C). We also analyzed the expression of NlMLP in 207
salivary glands by mRNA in situ hybridization. Hybridization signals were detected in 208
the A-follicles of principal glands but not in the salivary ducts or accessory glands 209
(Fig. 1D). 210
To confirm that NlMLP was secreted into rice tissue during feeding, we extracted 211
proteins from the leaf sheaths of plants following BPH feeding and analyzed them by 212
mass spectrometry. Four NlMLP peptides were detected in BPH-infested rice leaf 213
sheaths but not in non-infested rice (Fig. 1A), indicating that NlMLP was secreted into 214
the rice plants. 215
To determine the cellular localization of NlMLP in plant cells when transiently 216
expressed, we conducted localization experiments using rice protoplasts and N. 217
benthamiana leaf cells. When the NlMLP-GFP fusion protein was transiently 218
expressed in rice protoplasts, GFP fluorescence was detected only in the cytoplasm, 219
while control GFP fluorescence was detected in both the cytoplasm and nucleus (Fig. 220
1E). When the NlMLP-YFP fusion protein was transiently expressed in N. 221
benthamiana leaves via agroinfiltration, NlMLP localized to the cytoplasm 222
(Supplemental Fig. S3). 223
224
NlMLP is required for the feeding of BPHs on rice plants and for insect 225
performance 226
To elucidate the role of NlMLP in BPH, we synthesized double-stranded RNA (dsRNA) 227
from NlMLP and injected it into 4th instar BPH nymphs to mediate RNA interference 228
(RNAi). This treatment had a very strong silencing effect, reducing NlMLP transcript 229
levels significantly (~95%) on the first day after treatment compared to the levels in 230
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two control groups receiving either no injection or injection with dsGFP (P < 0.001 for 231
C and dsMLP from 1 to 6 days; P = 0.017 for dsGFP and dsMLP at 1 and 5 days; P = 232
0.016 for dsGFP and dsMLP at 2, 3 and 6 days; P = 0.015 for dsGFP and dsMLP at 4 233
days; Supplemental Fig. S4). The silencing was confirmed by RNA gel blot analysis 234
two days after injection (Supplemental Fig. S4). The treated BPH insects were allowed 235
to feed on TN1 rice plants. The survival rate of BPHs harboring a silenced NlMLP gene 236
was significantly lower (from 2 to 10 days following injection) than those of the two 237
control groups (P < 0.001 for C and dsMLP and P = 0.046 for dsGFP and dsMLP at 2 238
days; P < 0.001 for C and dsMLP and P = 0.001 for dsGFP and dsMLP at 10 days; 239
Fig. 2A). The cumulative mortality rate of BPHs injected with dsMLP, dsGFP, and 240
non-injected BPHs was 96%, 60%, and 52%, respectively, at 10 days after injection 241
(Fig. 2A). BPHs subjected to the NlMLP RNAi treatment also excreted significantly 242
less honeydew, a simple measurable indicator of BPH feeding activity, than the two 243
control groups (P < 0.001 for C and dsMLP; P = 0.003 for dsGFP and dsMLP; Fig. 244
2B), as well as having smaller weight gain values (P = 0.011 for C and dsMLP; P = 245
0.031 for dsGFP and dsMLP; Fig. 2C) and weight gain ratios (P = 0.023 for C and 246
dsMLP; P = 0.043 for dsGFP and dsMLP; Fig. 2D). Furthermore, silencing of NlMLP 247
reduced BPH virulence. Rice plants died in 7 days after infested by common BPH 248
insects or BPH insects injected with dsGFP, while those plants infested by BPH 249
insects injected with dsMLP still survived and grew normally (Supplemental Fig. S5). 250
These results indicate that silencing the NlMLP gene significantly reduced the feeding 251
and performance of BPHs on rice plants. 252
Feeding on rice plants expressing dsRNAs was previously shown to trigger RNA 253
interference of a target gene in BPH (Zha et al., 2011). We therefore transformed 254
BPH-susceptible rice plants with NlMLP-dsRNA and selected a T2 homozygous 255
dsMLP-transgenic line expressing NlMLP-dsRNA via qRT-PCR analysis 256
(Supplemental Fig. S6A). When 2nd instar BPHs were fed on dsMLP-transgenic plants, 257
their expression level of NlMLP was significantly (40%) lower than in BPHs fed on 258
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wild-type plants at 7 and 9 days after the start of exposure (P = 0.042 at 7 days; P = 259
0.045 at 9 days; Supplemental Fig. S6B). BPH survival and weight gain were also 260
significantly lower in insects fed on dsMLP-transgenic plants than in those fed on 261
wild-type plants from 7 to 10 days (P = 0.049 at 7 days; P = 0.046 at 8 days; P = 0.005 262
at 9 days; P = 0.008 at 10 days; Fig. 2E) and after 10 days (P = 0.023; Fig. 2F), 263
respectively. These results clearly show that NlMLP protein is essential for BPH 264
feeding and performance. 265
266
NlMLP is necessary for salivary sheath formation 267
NlMLP was found in both gelling saliva and watery saliva (Huang et al., 2016). To 268
further investigate the effects of NlMLP on feeding, we focused on salivary sheath 269
formation. First, we fed BPHs on an artificial diet in Parafilm sachets for 2 days and 270
analyzed their salivary sheaths by fluorescence microscopy and scanning electron 271
microscopy observation. The fluorescence microscopy analysis revealed that BPHs 272
subjected to the NlMLP RNAi treatment produced salivary sheaths that were 273
significantly shorter and less branched than those produced by the control BPHs 274
receiving either no injection or injection with dsGFP (Fig. 3, A and B). Moreover, the 275
structure of the sheaths was incomplete or predominantly amorphous, or gelling saliva 276
deposits at their stylet penetration sites were minimal, whereas those secreted by 277
control BPHs had complete, typical structures (Fig. 3, C-E). Second, we collected 278
stems from rice plants after BPH feeding and sectioned them to observe salivary sheath 279
morphology in planta. Most salivary sheaths in rice stems produced by the control 280
BPHs reached the phloem (Fig. 3, F and G), whereas most salivary sheaths produced 281
by NlMLP-silenced BPHs were shorter and failed to reach the phloem, instead stopping 282
in the rice epidermis or xylem (Fig. 3H). Together, these observations indicate that 283
NlMLP is necessary for salivary sheath formation. Silencing of NlMLP in BPHs 284
resulted in imperfect, short salivary sheaths, thus affecting phloem feeding. 285
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NlMLP induces plant cell death 287
The above findings clearly show that NlMLP is secreted into rice tissues (Fig. 1A) and 288
that it is localized to the cytoplasm of rice cells (Fig. 1E). To uncover the potential role 289
of NlMLP in the host plant, we transiently expressed NlMLP without the signal peptide 290
in rice protoplasts. We measured cell death, fluorescein diacetate (FDA) staining of the 291
protoplasts showed that the cell viability of protoplasts expressing NlMLP was 292
significantly lower than that of control protoplasts expressing GFP (P < 0.001; Fig. 4, 293
A and B). We also co-expressed NlMLP together with the luciferase (LUC) gene in rice 294
protoplasts. LUC activity was significantly lower in protoplasts co-expressing NlMLP 295
compared to the control co-expressing GFP (P < 0.001; Fig. 4C). Immunoblotting 296
confirmed that NlMLP and GFP were expressed properly in the rice protoplasts 297
(Supplemental Fig. S8A). These observations indicate that NlMLP expression triggers 298
cell death in rice protoplasts. To determine whether NlMLP-triggered cell death is 299
affected by the presence of BPH-resistance genes in rice, we performed similar LUC 300
assays after co-transfection of protoplasts with NlMLP and the genes Bph6, Bph9, and 301
Bph14. The LUC activity was still significantly lower in the presence of NlMLP than in 302
the GFP controls, regardless of the presence of resistance genes (P = 0.001 for Bph6; P 303
= 0.005 for Bph9; P = 0.004 for Bph14; Supplemental Fig. S7). Therefore, NlMLP 304
expression triggers cell death in rice protoplasts independently of these BPH-resistance 305
genes. 306
We further verified the ability of NlMLP to induce plant cell death by performing A. 307
tumefaciens-mediated expression of NlMLP in N. benthamiana leaves. INF1, an elicitin 308
secreted by Phytophthora infestans that induces HR cell death in Nicotiana plants 309
(Derevnina et al., 2016), was used as a positive control, while GFP was used as a 310
negative control. NlMLP, with or without YFP-HA tag, triggered marked cell death 311
(Fig. 4, D and E). Moreover, ion leakage was significantly higher from leaves 312
expressing NlMLP or INF1 than from the GFP-expressing controls (P = 0.002 for GFP 313
and INF1; P < 0.001 for GFP and NlMLP; Fig. 4F). Immunoblot analysis showed that 314
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GFP, INF1, and NlMLP proteins accumulated to comparable degrees in N. 315
benthamiana leaves (Supplemental Fig. S8B). However, the cell death symptoms 316
caused by INF1 and NlMLP is different. Leaves infiltrated with INF1 strain appeared 317
chlorosis in 4 days, and became severe necrosis accompanied by brown or black color 318
after 5 days. On leaves infiltrated with NlMLP, white or grey-white necrotic spots 319
appeared in 4 days and became bigger around the infiltrated site as time goes on 320
(Supplemental Fig. S9). We further investigated the quantity of NlMLP required for 321
the cell death symptoms. We set up different concentrations (OD600=0.005, 0.01, 0.02, 322
0.03, 0.04, 0.05, 0.08, 0.1, 0.2 and 0.3) of NlMLP strains to infect N. benthamiana 323
leaves, and found that cell death was caused by NlMLP strains in OD=0.01 or over 324
but was not when the OD is 0.005 (Supplemental Fig. S10A). Immunoblot detected 325
NlMLP protein in the leaves infected by in leaves infected with strains in OD 0.01 or 326
over, but not in 0.005 (Supplemental Fig. S10B). 327
To further characterize the physiological properties of the cell death induced by 328
NlMLP, we examined the effects of treatments that inhibit various potential cell 329
death-associated processes in rice protoplasts and N. benthamiana leaves. The 330
application of LaCl3 blocked the induction of cell death by NlMLP, suggesting that the 331
cell-death process mediated by NlMLP is dependent on a calcium signaling pathway 332
(Boudsocq et al., 2010) (Table 1). There was no difference in cell viability between 333
protoplasts incubated in the light or dark, indicating that the cell death process induced 334
by NlMLP is light-independent (Asai et al., 2000). Bcl-xl is an anti-apoptotic protein 335
(Chen et al., 2012). The expression of Bcl-xl in N. benthamiana leaves suppressed cell 336
death induced by subsequent NlMLP expression (Table 1). Our results indicate that cell 337
death induced by NlMLP shares some common properties with cell death induced by 338
BAX and INF1. MAPK cascades play important roles in defense-related signal 339
transduction (Yang et al., 2001). MEK2 is a MAPK kinase that acts upstream of 340
SA-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) and 341
controls multiple defense responses to pathogen invasion (Yang et al., 2001). When we 342
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silenced MEK2 in N. benthamiana plants via VIGS (P = 0.006; Fig. 4I), 343
NlMLP-triggered cell death was significantly reduced in MEK2-silenced plants (Fig. 344
4H), but not in control plants (Fig. 4G). However, the presence of INF1, which triggers 345
cell death independently of MEK2 (Takahashi et al., 2007), clearly caused necrosis in 346
MEK2-silenced plants (Fig. 4H). Therefore, NlMLP-triggered cell death is associated 347
with MEK2-dependent MAPK cascades. 348
349
NlMLP triggers plant defense responses 350
Callose deposition is used as a marker for plant basal defense responses and participates 351
in plant defenses against phloem sap ingestion by insects (Hann and Rathjen, 2007; 352
Hao et al., 2008). Thus, to determine whether NlMLP activates defense responses in 353
planta, we expressed NlMLP in N. benthamiana leaves and investigated callose 354
deposition by aniline blue staining. Many more callose spots were present in 355
NlMLP-expressing leaves (47.0 per infiltration) than in GFP-expressing leaves (3.5 per 356
infiltration) (P = 0.001; Fig. 5A). Moreover, NlMLP induced transcriptional activation 357
of the pathogen resistance (PR) genes NbPR3 (P = 0.143 at 24h; P = 0.002 at 48h) and 358
NbPR4 (P = 0.002 at 24h; P = 0.002 at 48h), but not NbPR1 (P = 0.625 at 24h; P = 359
0.180 at 48h), within 48 h of infection (Fig. 5B). The upregulation of genes encoding 360
acidic NbPR1 protein is a characteristic feature of the activated SA-signaling pathway, 361
while the induction of genes encoding basic NbPR3 and NbPR4 proteins is associated 362
with JA-dependent defense responses (Zhang et al., 2012; Naessens et al., 2015). Thus, 363
NlMLP appears to induce defense responses mediated by the JA signaling pathway, 364
thereby promoting the production of PR proteins and the biosynthesis of cell 365
wall-reinforcing callose. 366
367
The functional motif is located in the C-terminus of NlMLP 368
NlMLP showed no sequence similarity to any known cell death-inducing effector. To 369
delineate the functional domains of NlMLP, we assayed the ability of N-terminal and 370
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C-terminal deletion mutant proteins to trigger cell death in N. benthamiana leaves (Fig. 371
6). The N-terminal deletion mutant M428-728
strongly triggered cell death, but the 372
C-terminal deletion mutants M32-319
and M319-428
did not. Moreover, M428-674
triggered 373
cell death less strongly than did M428-728
, and the M674-728
mutant did not induce cell 374
death at all. These findings suggest that the 428-674 amino-acid fragment is required 375
for triggering cell death and that the 674-728 amino-acid fragment might promote this 376
effect. We also found that the 428-674 fragment was required for the expression of 377
NbPR4 (P = 0.001 for GFP and M1-728
; P = 0.002 for GFP and M32-728
; P = 0.008 for 378
GFP and M319-728
; P < 0.001 for GFP and M428-728
; P = 0.129 for GFP and M674-728
; P 379
= 0.007 for GFP and M428-674
; P = 0.209 for GFP and M319-428
; P = 0.656 for GFP and 380
M32-319
; Fig. 6). Immunoblot analysis showed that the mutant proteins accumulated to 381
comparable levels in N. benthamiana leaves (Supplemental Fig. S8C). 382
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16
DISCUSSION 383
This is the first report of a planthopper salivary protein that plays important roles in 384
feeding and interactions with the host plant. We identified a N. lugens mucin-like 385
protein, NlMLP, which is highly expressed in the salivary glands of BPHs and secreted 386
into rice tissue during BPH feeding. NlMLP is necessary for the probing of rice plants 387
by BPHs to obtain phloem sap for insect survival. BPH feeding was inhibited and insect 388
performance was significantly reduced when NlMLP expression was knocked-down 389
(Fig. 2). Furthermore, the salivary sheaths produced by NlMLP-silenced BPHs were 390
shorter and less branched than those of control BPHs fed on both an artificial diet and 391
rice tissue. The sheaths had incomplete structures and were predominantly 392
amorphous. 393
Mucin-like proteins have been identified in various organisms; some mucins are 394
involved in controlling mineralization (Boskey, 2003) and are associated with 395
processes including nacre formation in mollusks (Marin et al., 2000), calcification in 396
echinoderms (Boskey, 2003), and bone formation in vertebrates (Midura and Hascall, 397
1996). Based on our current results, mucin appears to be an essential component of 398
the BPH salivary sheath. Notably, the mucin-like protein NlMLP is rich in serine, 399
which provides attachment sites for carbohydrate chains that participate in the 400
formation of large extracellular aggregates (Korayem et al., 2004), thus functioning in 401
the formation of salivary sheaths, which support stylet movements and exploration of 402
the host plant tissue. Therefore, reductions in the levels of NlMLP may prevent the 403
construction of complete sheaths. The immediate consequence of this reduction in 404
NlMLP-RNAi insects is that salivary sheaths formed in rice plants do not reach into the 405
sieve tube, which likely accounts for the reduction in phloem feeding and performance 406
of these insects on rice plants. 407
In aphids, the salivary protein SHP contributes to the solidification of gelling saliva 408
and sheath formation, partly through the formation of disulfide cystine bonds (Carolan 409
et al., 2009; Will and Vilcinskas, 2015). Mucins can form disulfide-dependent soluble 410
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17
dimers and multimeric insoluble gels through cross-linking of cysteines (Axelsson et 411
al., 1998). Cysteine residues in NlMLP might behave in a similar fashion and strongly 412
contribute to the formation of the polymeric matrix during sheath hardening via the 413
formation of intermolecular disulfide bonds. NlMLP might also function in the growth 414
and development of BPHs. Silencing of NlMLP affected insect development, which in 415
turn reduced salivary sheath formation, feeding, and performance. 416
Plants usually detect molecules emitted by parasites to trigger defense responses. 417
When BPHs feed on phloem sap, their saliva is secreted into rice tissue, which contains 418
bioactive components involved in inducing the expression of defense genes. We 419
demonstrated that NlMLP is one such bioactive component. Mucin-like proteins have 420
also been detected in fungal pathogens. The surfaces of many parasites, including the 421
protozoan parasite Trypanosoma cruzi (Buscaglia et al., 2006), the fish pathogen T. 422
carassii (Lischke et al., 2000), and the potato pathogen Phytophthora infestans 423
(Gornhardt et al., 2000) are covered in mucins, which participate in interactions with 424
host cells during the invasion process (Buscaglia et al., 2006; Larousse et al., 2014). 425
Similarly, NlMLP is highly expressed in salivary glands and is secreted into the plant. 426
We found that NlMLP expression triggered cell death in rice protoplasts and N. 427
benthamiana leaves (Fig. 4), as well as plant immunity responses, including the 428
induction of PR gene expression and callose synthesis in leaves (Fig. 5). NlMLP 429
molecules on the surface of the salivary sheath, representing an essential component 430
of this structure, can be detected as signal of BPH feeding by host cells and evoke 431
defense responses. NlMLP in watery saliva may play the same role as well. Our 432
results indicate that the functional portion of NlMLP is located at its C-terminus. The 433
presence of a 428-674 amino-acid fragment was sufficient for triggering cell death and 434
inducing the expression of NbPR4. Mucin-like proteins are also detected in other 435
piercing-sucking insects such as leafhopper (Hattori et al., 2015) and several mosquito 436
species (Das et al., 2010). The wide taxonomic range of hosts in which NlMLP can 437
trigger cell death suggests that NlMLP may be recognized by a conserved protein found 438
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18
in many plants. The plant receptor that recognizes NlMLP remains to be identified. 439
The defense reaction elicited by NlMLP shares common features with immune 440
responses which shown by well-known effectors and pathogen-associated molecular 441
patterns (PAMPs). NlMLP might be an elicitor that involved in the PTI process. It 442
may be recognized by plant PRRs which triggers plant defensive responses. NlMLP 443
triggers cell death, a common phenomenon in effector-triggered immune responses. 444
Ca2+
is a well-known secondary signal in eukaryotes. The early defense response to 445
BPH in rice involves Ca2+
influx, which is a common early plant response triggered 446
by insect feeding (Hao et al., 2008; Hogenhout and Bos, 2011; Bonaventure, 2012). 447
Our results indicate that the cell death process induced by NlMLP is dependent on 448
Ca2+
influx. Moreover, cell death induced by NlMLP is not dependent on light and is 449
suppressed by the anti-apoptotic protein Bcl-xl. NlMLP-triggered cell death requires 450
the MEK2 MAP kinase signal transduction pathway. MEK2 is a common component in 451
MAP kinase pathways required for HR followed by certain Avr-R interactions in 452
tomato and tobacco (Morris, 2001; Del Pozo et al., 2004; King et al., 2014). NbMEK2 453
is also required for cell death triggered by the Phytophthora sojae RxLR effector 454
Avh241 in N. benthamiana (Yu et al., 2012). Given the broad range of plants 455
responding to NlMLP and the dependence of these responses on the MEK2 pathway, 456
we speculate that plants respond to NlMLP may via a conserved upstream component 457
of plant signaling pathways. The expression of NlMLP induced the JA pathway marker 458
genes NbPR3 and NbPR4. NbPR3 is a chitinase gene associated with JA-dependent 459
defenses that is induced by elicitors (Naessens et al., 2015), while NbPR4 encodes a 460
hevein-like chitinase that is also primarily associated with JA responses (Kiba et al., 461
2014). The findings suggest that defense responses triggered by NlMLP are associated 462
with the JA signaling pathway. NlMLP expression in N. benthamiana leaves also 463
induced callose deposition. Callose deposition is a useful mechanism conferring plant 464
immunity to insects and pathogens (Luna et al., 2011). In the interaction of rice and 465
BPH, callose deposition on the sieve plates occludes sieve tubes, thereby directly 466
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19
inhibiting continuous feeding by BPHs (Hao et al., 2008). 467
Nevertheless, BPH can successfully attack most rice cultivars and even results in 468
hopper burn. This success is thought because of the evolved ability of BPH to suppress 469
or counteract rice defense responses triggered by NlMLP and other elicitors (Walling, 470
2008 Kaloshian and Walling, 2016). Hemipterans saliva is a complex mixture of 471
biomolecules with potential roles in overcoming plant immune responses and enables 472
hemipterans to manipulate host responses to their advantage (Miles 1999; Kaloshian 473
and Walling, 2016). One example is that BPH can decompose the deposited callose and 474
unplug sieve tube occlusions by activating b-1,3-glucanase genes and thereby facilitate 475
the continuous phloem-feeding in rice plants (Hao et al., 2008). To date, some effectors, 476
including C002, Me10, Me23, Mp1, Mp2, and Mp55, have been identified by assays in 477
planta overexpression and RNAi in aphids. These effectors contribute to aphid survival 478
and suppressing host defense (Mutti et al., 2008; Bos et al., 2010; Atamian et al., 2013; 479
Pitino and Hogenhout, 2013; Elzinga et al., 2014). The effectors may target any 480
component in pattern-triggered immunity or host cell trafficking pathway (Derevnin et 481
al., 2016; Kaloshian and Walling, 2016; Rodriguez et al., 2017). It is likely that potent 482
effectors in BPH also target such cell processes in rice and enable BPH to successfully 483
feed on rice plants, which induces cascade reactions termed the BPH-feeding cascade, 484
and results in the death of susceptible rice varieties (Cheng et al., 2013). In contrast, in 485
resistant rice the resistance gene induces a strong defense response that inhibits insect 486
feeding, growth, and development, and enables the plant to grow normally. 487
In summary, our results indicate that NlMLP secreted by BPHs into rice plants plays 488
dual roles as a component in feeding sheath formation and activating plant defense 489
responses. The defense responses induced by NlMLP in plant cells are related to Ca2+
490
mobilization and the MEK2 MAP kinase and JA signaling pathways. Further studies 491
are needed to identify the target of NlMLP in rice cells and BPH effectors that suppress 492
rice defense responses to further explore the interaction mechanisms between plants 493
and planthoppers. 494
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20
MATERIALS AND METHODS 495
Plant materials and insects 496
Wild-type and transgenic rice (Oryza sativa) plants were grown in the experimental 497
fields at Wuhan University Institute of Genetics, China under routine management 498
practices. Brown planthopper biotype 1 insect populations were reared on 499
one-month-old plants of the susceptible rice variety TN1 under controlled 500
environmental conditions (26 ± 0.5°C, 16 h light/8 h dark cycle). 501
502
Tanscriptome analysis of BPH salivary glands 503
BPH adult females were dipped in 70% ethanol for several seconds and washed in 504
0.85% NaCl solution. Salivary glands were dissected in phosphate buffer saline 505
solution (pH 7.4). This was accomplished by pulling the head off of the thorax with 506
forceps and carefully removing the salivary glands that emerged from the distal region 507
of the severed head. A total of 2,000 salivary glands were dissected and directly dipped 508
into 300 µL RNAiso Plus (Takara) for RNA preparation. The poly (A) RNA was 509
isolated, broken into smaller fragments (200-700 bp), and reverse-transcribed to 510
synthesize cDNA for Illumina HiSeq™ 2000 sequencing. After stringent quality 511
assessment and data filtering, 40,000,000 reads were selected as high-quality reads 512
(after removing adapters and low-quality regions) for further analysis. A set of 513
12,668,039 high-quality reads with an average length of 400 bp was assembled, 514
resulting in 59,510 contigs. The contigs were assembled into 31,645 unigenes, 515
consisting of 3960 clusters and 27,685 singletons. All unigene sequences were aligned 516
by Blastx to protein sequences in databases including nr, Swiss-Prot, KEGG, and COG, 517
followed by sequences in the non-redundant NCBI protein database (with e<10-5
in 518
both cases). 519
SignalP 3.0 was used to predict the presence of signal peptides. To predict 520
transmembrane domains, each amino acid sequence with a signal peptide was 521
submitted to the TMHMM Server v. 2.0. 522
523
Cloning and sequencing of NlMLP cDNA 524
The cDNA sequences of NlMLP were obtained from salivary-gland transcriptomes 525
of BPHs. To obtain the full-length sequence of each truncated sequence from BPHs, 5’ 526
and 3’ RACE amplification were performed using a 5’-Full RACE Kit and 3’-Full 527
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21
RACE Core Set Ver. 2.0 (Takara) following the manufacturer’s instructions. For 528
5’-RACE, gene-specific primers were designed based on sequencing data, and an 529
external reverse primer (5’-RACE-EP) and a nested primer (5’-RACE-NP) were used. 530
For 3’-RACE, the cDNA was then amplified by nested PCR with the external forward 531
primer (3’-RACE-EP) and the nested forward primer (3’-RACE-NP) (Table S2) with 532
La Taq DNA polymerase (Takara). Purified PCR products were ligated to the 533
pMD18-T simple vector (Takara) and positive colonies were sequenced. 534
535
Proteomics analysis of rice leaf sheathes 536
The TN1 rice plants were grown in pots (8 cm in diameter and 15 cm in height). At the 537
fifth leaf stage, 2nd and 3rd-instar BPH nymphs were released onto the plants at 20 538
insects per plant. The leaf sheaths of BPH-infested plants and non-infested control 539
plants were collected after 72 h, immediately frozen in liquid nitrogen, and stored at 540
-80°C. Protein extraction and digestion, iTRAQ labeling, strong cation exchange 541
chromatography (SCX) fractionation, and LC−MS/MS proteomic analysis were 542
performed according to the manufacturer’s protocols using an iTRAQ Reagent 8 Plex 543
Kit (SCIEX). A TripleTOF 5600 (AB SCIEX, Concord, ON), fitted with a NanoSpray 544
III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New 545
Objectives, Woburn, MA), was used for MS/MS. Protein identification was performed 546
using Mascot 2.3.02 (Matrix Science, London, UK) against the transcriptomic database 547
of N. lugens salivary glands (containing 18,099 protein-coding sequences). 548
549
Expression analysis of NlMLP 550
The temporal and spatial expression patterns of NlMLP were investigated by 551
qRT-PCR as follows. For spatial expression pattern analysis, salivary glands, guts, fat 552
bodies, and carcasses (i.e., samples consisting of all parts remaining after the removal 553
of the salivary gland, gut, and fat body) were dissected from BPH adults under a 554
stereomicroscope. Total RNA was isolated from each type of tissue using RNAiso 555
Plus (Takara). For temporal expression pattern analysis, total RNA was isolated from 556
BPH at various developmental stages including eggs, 1st to 5th instar nymphs, female 557
adults, and male adults. First-strand NlMLP cDNA was obtained from all samples by 558
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22
reverse transcription using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara) 559
according to the manufacturer’s instructions, followed by amplification by qRT-PCR 560
using a Bio-Rad CFX-96 Real-Time PCR system with an iTaq Universal SYBR 561
Green Supermix Kit (Bio-Rad, USA) and gene-specific primers qNlMLP-F and 562
qNlMLP-F (Table S2). As an endogenous control to normalize expression levels with 563
average threshold cycle (Ct) numbers, a partial fragment of the BPH actin gene was 564
amplified with primers qNlActin-F and qNlActin-R (Table S2). A relative quantitative 565
method (ΔΔCt) was applied to evaluate the variation in expression among samples. 566
567
Fluorescence in situ hybridization of BPH salivary glands. 568
The expression patterns of NlMLP within the salivary glands were examined by in 569
situ mRNA localization and confocal microscopy, as follows. BPHs were anesthetized 570
on ice and their salivary glands were dissected and fixed in 4% formaldehyde in PTw 571
(phosphate buffered saline + 0.1% Tween) overnight at 4°C. The fixed glands were 572
treated with 5 mg/mL Proteinase K for 10 minutes and subjected to various 573
hybridization steps (Villarroel et al., 2016). Briefly, the glands were prehybridized in 574
hybridization buffer for 1 h at 52°C, incubated in fresh hybridization buffer and probe 575
overnight at 52°C, and subjected to six 25 min washes at 52°C with wash buffer. After 576
washing at room temperature with PBTw, the glands were incubated for 3-5 h at 4°C in 577
the dark in a 1:200 dilution of anti-digoxigenin-fluorescein (Fab fragments, Roche) in 578
PBTw. The samples were then washed with PTw for 20 min and mounted in 70% 579
glycerol in PTw. Fluorescence images were obtained under an Olympus Fluoview 580
FV1000 confocal laser-scanning microscope (Olympus Corporation, Japan). 581
582
DsRNA synthesis and injection to trigger RNA interference (RNAi) in insects 583
The DNA template for NlMLP dsRNA (dsMLP) synthesis was obtained using primer 584
pair dsMLP-F and dsMLP-R. DsMLP was synthesized using a MEGAscript T7 High 585
Yield Transcription Kit (Ambion) according to the manufacturer’s instructions. The 586
amplified NlMLP fragment (500 bp) was confirmed by sequencing. The primers 587
dsGFP-F and dsGFP-R, which were designed based on the GFP (green fluorescent 588
protein) gene template, were used to synthesize dsGFP as a negative control in the 589
RNAi experiments. The dsRNA was purified by phenol chloroform extraction and 590
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23
resuspended in nuclease-free water at a concentration of 5 µg/µL. 591
Fourth-instar nymphs were anesthetized with carbon dioxide for approximately 20 592
sec until they were unconscious and placed on 2% agarose plates with their abdomens 593
facing up. A 46 nL volume of dsMLP or dsGFP was injected into each insect at the 594
junction between the prothorax and mesothorax using a microprocessor-controlled 595
Nanoliter 2010 injector (World Precision Instruments, USA). The injected BPHs were 596
reared on rice cv. TN1 plants. 597
598
Bioassay of BPHs after dsRNA injection 599
The survival rates of BPHs injected with dsGFP or dsMLP and the non-injected 600
controls were determined as follows. Pots (diameter 7 cm, height 9 cm), each 601
containing a single one-month-old TN1 rice plant grown and maintained as described 602
above, were individually covered with plastic cages into which 10 injected BPH 603
nymphs were released. The number of surviving BPH nymphs on each plant was 604
recorded daily for 10 days. The experiment was repeated five times. 605
BPH growth rates were analyzed using newly emerged brachypterous females 606
(within one day of emergence), Parafilm sachets (2×2.5 cm), and one-month-old TN1 607
rice plants grown and maintained as described. After weighing the insects and sachets, 608
the insects were placed into the sachets, which were then attached to the plants (with 609
one BPH per sachet and one sachet per plant). After 72 h, each insect and Parafilm 610
sachet was re-weighed, and the changes in weight of the BPH and sachet were defined 611
as BPH weight gain and honeydew weight, respectively. The BPH weight gain ratio 612
was calculated as the change in weight relative to the initial weight. The experiment 613
was repeated 10 times per group, and the experiments were conducted five times. 614
615
Honeydew excretion on filter paper. 616
Honeydew excretion on filter paper was measured as previously described (Du B et 617
al., 2009). One-month-old rice cv. TN1 plants grown in pots (diameter 7 cm, height 9 618
cm, one plant per pot) as described were covered with an inverted transparent plastic 619
cup placed over filter paper resting on a plastic Petri dish. Two days after injection, 620
the BPHs were placed into the chamber. After 2 days of feeding, the filter paper was 621
collected and treated with 0.1% ninhydrin in acetone solution. After 30 min of oven 622
drying at 60°C, the honeydew stains appeared violet or purple due to their amino acid 623
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24
content. The area of the ninhydrin-positive deposits was measured with Image J 624
software. The experiment was repeated three times. 625
626
Development of dsMLP-transgenic rice and the BPH bioassay 627
To constitutively express NlMLP-dsRNA in rice, a 500 bp template fragment (the same 628
as the target sequence used for micro-injection) and stuffer sequence fragment (a PDK 629
intron) were used to generate a hairpin RNAi construct as previously described (Zha et 630
al., 2011). The construct was inserted into binary vector pCXUN (accession no. 631
FJ905215) under the control of the plant ubiquitin (ubi) promoter. The construct was 632
transformed into rice cv. Kasalath (an indica rice variety susceptible to BPH and 633
amenable to gene transformation) using an Agrobacterium-mediated method. 634
Integration of the foreign DNA in T0 plants was confirmed by PCR and DNA gel blot 635
analysis. The plants were cultivated, and T2 seeds were collected for the bioassay. 636
The survival rates of BPHs on transgenic and WT plants (Kasalath) were determined 637
by releasing 20 2nd instar nymphs onto each plant. The number of surviving BPHs was 638
recorded daily for 10 days. The experiment was repeated five times. NlMLP 639
expression in BPHs was evaluated by qRT-PCR. BPH weight was measured on a 640
microbalance after 10 days of feeding. 641
642
Observation of salivary sheaths on Parafilm and in planta 643
To observe the morphology of the salivary sheaths, BPHs were fed on an artificial diet 644
(D-97) consisting of amino acids, vitamins, inorganic salts, and sucrose (Fu et al., 645
2001) using plastic cylinders (4.0 cm long and 2.5 cm in diameter) as feeding 646
chambers. Sachets formed from two layers of stretched Parafilm M (~4-times the 647
original area), each containing 200 µL portions of D-97, were placed at one end of the 648
chamber. The opposite end of the chamber was covered with a piece of nylon mesh 649
after the test insects had been introduced. Insects could feed by piercing through the 650
inner Parafilm layer of the diet sachets, leaving salivary sheaths in the sachets. To 651
assess the effects of dsRNA injection on salivary sheaths, two days after injection, 652
nymphs were individually placed in the chambers and allowed to feed for 48 h. The 653
inner Parafilm layers were then placed on a microscope slide and the salivary sheaths 654
were counted under a BX51 light microscope (Olympus). Sets of 10 655
dsNlMLP-injected, dsGFP-injected, and non-injected control BPHs were used per 656
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25
experiment, and the experiment was repeated three times. 657
To obtain samples for scanning electron microscopy (SEM), the inner Parafilm 658
layer was placed on a microscope slide and salivary sheaths were identified under a 659
light microscope (Olympus BX51). Regions of interest were labeled and the Parafilm 660
was cut with a scalpel. The salivary sheaths from the Parafilm were attached to a 661
sample holder, coated with gold, and observed under an S-3400N SEM (Hitachi). Five 662
replicates were prepared per treatment, and 20 randomly chosen salivary sheaths were 663
observed. 664
The dsRNA-treated BPHs were reared on one-month-old TN1 rice seedlings for 48 h, 665
and the leaf sheaths were collected and used for paraffin sectioning. One-month-old 666
rice cv. TNI plants (grown and maintained as described) were infested with a set of 10 667
dsMLP-injected, dsGFP-injected, or non-injected BPHs. The leaf sheaths produced by 668
the insects were collected, fixed in 4% paraformaldehyde, dehydrated, embedded in 669
paraffin, and cut into 10 mm thick sections with a microtome. The sections were 670
mounted on microscope slides and stained with 0.25% Coomassie Blue staining 671
solution for 10 min. The slides were dewaxed, rehydrated, examined, and photographed 672
under a light microscope. 673
674
675
Viability assay using rice protoplasts 676
Rice (cv. 9311 an indica rice variety susceptible to BPH) protoplasts were isolated 677
from 10-day-old plants as previously described (Zhang et al., 2011). The cell viability 678
and luciferase assays were conducted using rice protoplasts as described (Zhao et al., 679
2016). Briefly, for the cell viability assay, protoplasts were transfected with the 680
indicated plasmids for 20 h and stained with 220 μg/mL FDA. For the luciferase 681
assays, protoplasts were co-transfected with the reporter Renilla luciferase gene 682
(RLUC) and another construct carrying GFP or NlMLP. Luciferase activity was 683
measured 40 h after transfection using a luciferase assay system (Promega). 684
685
A. tumefaciens infiltration assays of N. benthamiana leaves 686
A sequence of interest was amplified by PCR. The PCR products and the PUC19 vector 687
were both digested by Not I and Asc I and bound by DNA ligase to create entry vectors. 688
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26
Using LR Clonase reaction enzyme mix (Invitrogen), the target sequence was 689
recombined into the destination vector pEarleyGate 101 (with the YFP-HA epitope tag) 690
or pEarleyGate 100 (with no epitope tag). Constructs were introduced into A. 691
tumefaciens strain GV3101 by electroporation. Recombinant strains were cultured in 692
Luria-Bertani medium supplemented with 50 µg/mL kanamycin and 50 μg/mL 693
rifampicin, harvested, washed three times in infiltration medium (10 mM MES pH 5.6, 694
10 mM MgCl2, 150 µM acetosyringone), and resuspended in infiltration medium to an 695
OD600 of 0.3. The resuspended recombinant strains were incubated for 2 h at room 696
temperature and infiltrated into the leaves of 4- to 6-week-old N. benthamiana plants 697
(grown in a greenhouse at 25: 20°C under a 16 h light: 8 h dark cycle) through a nick 698
created using a needleless syringe. Symptom development in N. benthamiana was 699
monitored visually 3-8 d after infiltration. To test the difference of cell death symptoms 700
caused by INF1 and NlMLP, 12 pieces of N. benthamiana leaves were injected. 701
Symptom development in N. benthamiana was continued monitored visually every day 702
after infiltration. Four independent biological replicates were conducted and got 703
similar results. 704
To test the induction of plant defense-related gene expression by NlMLP, RNA was 705
extracted from N. benthamiana leaves 48 h after infiltration. SYBR green qRT-PCR 706
assays were performed to determine gene expression levels as previously described. 707
Three independent biological replicates were conducted for each experiment. 708
Agroinfiltrated N. benthamiana leaves were harvested at 48 h post inoculation. Total 709
protein extracts were prepared by grinding 400 mg of leaf tissue in 1 mL extraction 710
buffer (20 mM Tris-Cl, 1% SDS, 5 mM EDTA) in the presence of 10 mM DTT. The 711
samples were shaken on a vortex for 30 s and centrifuged at 800 g for 10 min at 4°C. 712
The resulting supernatant was used for immunoblot analysis. 713
Cell death was assayed by measuring ion leakage from leaf discs. Four leaf discs of 714
equal dimension (10 mm) were placed into 5 mL of distilled water. The first set was 715
incubated at room temperature overnight and its conductivity (C1) recorded using a 716
conductivity meter (FiveGO-FG3). The second set was boiling and its conductivity (C2) 717
recorded after cooling. Relative electrolyte leakage [(C1/C2) x 100] was calculated. 718
The experiments were carried out four times. 719
Callose deposition in infiltrated leaf disks 48 h after infiltration was visualized after 720
aniline blue staining following published methods (Naessens et al., 2015). Briefly, 721
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27
dissected leaf disks were destained by successive washes in 70%, 95%, and 100% 722
EtOH for two hours per wash, washed twice with ddH2O, and incubated in aniline blue 723
solution (70 mM KH2PO4, 0.05% [w/v] aniline blue, pH 9) for 1 h. Leaf discs were 724
mounted in 80% glycerol and observed under a fluorescence microscope (FV1000, 725
Olympus). After collecting callose images, fluorescence in the images was quantified 726
with ImageJ software. Each experiment was repeated three times independently. 727
728
Virus-induced gene silencing of MEK2 in N. benthamiana plants. 729
Silencing of NbMEK2 in N. benthamiana was performed by Potato virus X (PVX) 730
VIGS as described by Sharma et al. (2003). The NbMEK2 sequence (267 bp from the 3′ 731
terminus) was inserted into the PVX vector in the antisense direction to generate 732
PVX:MEK2. The construct containing the insert and the empty vector PVX were 733
transformed into A. tumefaciens strain GV3101. Bacterial suspensions were applied to 734
the undersides of N. benthamiana seedlings (~20 days) using a 1 mL needleless syringe. 735
The plants exhibited mild mosaic symptoms 3-4 weeks after inoculation. MEK2 gene 736
expression was measured by qRT-PCR, and plants in which silencing was established 737
were subjected to further analysis. 738
739
Cell death inhibition assays. 740
The effects of the calcium channel inhibitor LaCl3 and the human anti-apoptotic gene, 741
Bcl-xl, on cell death (under dark conditions) were determined as previously described4. 742
For the rice protoplast transient expression assay and agroinfiltration into N. 743
benthamiana leaves, rice protoplast or A. tumefaciens cultures were resuspended in 744
LaCl3 at a final concentration of 1 mM. Transient expression assays were performed 745
under dark conditions to determine whether cell death induced by NlMLP is light 746
dependent. Isolated protoplasts and N. benthamiana plants were incubated in the dark 747
for 30 minutes before transfection and maintained in the dark after transfection. Bcl-xl 748
was cloned into binary vector pGR107 and introduced into A. tumefaciens strain 749
GV3101. NlMLP or the cell death-inducing gene BAX or INF1 was expressed in N. 750
benthamiana leaves 24 h after introduction of the Bcl-xl expression vector or the empty 751
control vector via agroinfiltration. 752
753
Statistical analysis 754
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28
Data were compared using a Student’s t-test or a Tukey honest significant difference 755
test using PASW Statistics v18.0. 756
757
Supplemental Materials 758
Supplemental Fig. S1 Clusters of orthologous group (COG) classification of 759
sequences in the BPH salivary gland transcriptome. 760
Supplemental Fig. S2 Gene ontology (GO) classification of sequences in the BPH 761
salivary gland transcriptome. 762
Supplemental Fig. S3 NlMLP is localized to the cytoplasm in plant cells. 763
Supplemental Fig. S4 Confirmation of the effect of RNA interference on NlMLP 764
expression in BPHs. 765
Supplemental Fig. S5 Virulence of BPHs on rice plants. 766
Supplemental Fig. S6 Relative expression of NlMLP in transgenic rice (A) and BPH 767
insects (B). 768
Supplemental Fig. S7 NlMLP induces cell death in rice irrespective of the presence of 769
BPH-resistance genes (Bph6, Bph9, and Bph14). 770
Supplemental Fig. S8 Immunoblot analysis of proteins in rice protoplasts and N. 771
benthamiana. 772
Supplemental Fig. S9 Symptoms of cell death caused by INF1 and NlMLP in N. 773
benthamiana leaves. 774
Supplemental Fig. S10 Quantitative estimate of NlMLP required for plant symptoms. 775
776
Table S2 List of PCR primers used in this study. 777
Table S3 List of qRT-PCR primers used in this study. 778
779
Supplemental Methods S1 Tanscriptome analysis of BPH salivary glands. 780
Supplemental Methods S2 Gene silencing analysis by qRT-PCR. 781
Supplemental Methods S3 Gene silencing analysis by RNA gel blotting. 782
Supplemental Methods S4 Virulence of BPHs on rice plants. 783
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29
Supplemental Methods S5 Determining the quantity of NlMLP required for the cell 784
death symptoms. 785
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30
Table 1. Results of inhibition assays of cell death in rice protoplasts and N. 786
benthamiana leaves. 787
788
Gene Treatment
LaCl3a Dark
b Bcl-xl
c
LUC activity
in RPd
Cell
death in
NBLe
LUC activity
in RP
Cell
death in
NBL
LUC
activity in
RP
Cell
death in
NBL
GFP 1.00± 0.19 0/8 1.00± 0.34 0/8 ND 0/8
NlMLP 0.74± 0.17 1/8 0.14± 0.02* 8/8 ND 0/8
BAX 0.66± 0.21 3/8 0.09± 0.02* 7/8 ND 0/8
INF1 0.92± 0.34 3/8 0.38± 0.11* 7/8 ND 6/8
a LaCl3 was applied to protoplasts immediately after transfection at a final 789
concentration of 1 mM. For agroinfiltration of N. benthamiana leaves, LaCl3 was 790
added to resuspended A. tumefaciens cultures at a final concentration of 1 mM. 791
b Rice protoplasts and N. benthamiana leaves were incubated in the dark for 30 minutes 792
before transfection and maintained in the dark after transfection. 793
c N. benthamiana leaves were pre-infiltrated with A. tumefaciens cells harboring the 794
Bcl-xl expression vector. 795
d LUC activity in rice protoplasts, data represent means ± SE of three repeats. 796
Asterisks after the number indicate significant differences compared with the 797
corresponding GFP (*, P < 0.05, Student's t-test). 798
e Number of cell death sites/ the total infiltrated leaves of N. benthamiana. 799
ND = not determined 800
801
Figure legends 802
Figure 1. Molecular characterization of NlMLP. 803
A, Amino acid sequence of NlMLP. The solid underline indicates the signal peptide 804
predicted by SignalP. The asterisk (*) indicates the stop codon. The shaded amino acid 805
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31
residues indicate the peptides detected in BPH-infested rice tissue by LC-MS/MS 806
analyses. Ser is shown in red. The repeat region is boxed. 807
B and C, Expression patterns of NlMLP at different developmental stages (B) and in 808
different tissues (C). Relative levels of NlMLP expression at the indicated 809
developmental stages (1st to 5
th, 1
st to 5
th instar; F, female adult; M, male adult) and in 810
different tissues (salivary gland, gut, fat body, and remaining carcass) normalized 811
against β-actin gene expression, as determined by qRT-PCR. Data represent means ± 812
SE of three repeats. N = 30. Different letters above the bars indicate significant 813
differences, as determined by a Tukey honest significant difference test (P < 0.05). 814
D, mRNA in situ hybridization of NlMLP in salivary glands. Signals from 815
anti-DIG-FITC (shown in green) bound to a DIG-labeled antisense riboprobe used for 816
hybridization to NIMLP transcripts were detected by confocal laser-scanning 817
microscopy. PG, principal gland. AG, accessory gland. Scale bar = 100 μm. 818
E, NlMLP localizes to the cytoplasm of rice cells when transiently expressed. GFP or 819
NlMLP-GFP fusion protein was expressed in rice protoplasts by PEG-mediated 820
transformation. Confocal laser-scanning microscopy was used to investigate fusion 821
protein distribution 16 h after transformation. Scale bar = 5 μm. 822
823
Figure 2. Effects of NlMLP silencing on BPH feeding and performance. 824
A, The survival rates of BPH insects after injection were monitored daily. The survival 825
rates of BPHs injected with dsMLP was significantly reduced compared to BPHs with 826
no injection and those injected with dsGFP after 2 days of treatment (Student’s t-test). 827
C, BPHs with no injection; dsGFP, BPHs injected with GFP-dsRNA; dsMLP, BPHs 828
injected with NlMLP-dsRNA. The experiment was repeated five times, with 10 BPHs 829
per replicate. Data represent means ± SD of five repeats. 830
B, Honeydew excretion by BPH insects on filter paper. The intensity of the honeydew 831
color and area of the honeydew correspond to BPH feeding activity. The experiment 832
was repeated three times, with 10 filter papers per replicate. Data represent means ± SE 833
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32
of three repeats. Different letters above the bars indicate significant differences, as 834
determined by a Tukey honest significant difference test (P < 0.05). 835
C and D, BPH weight gain (C) and BPH weight gain ratio (D) of BPHs after injection 836
with dsGFP or dsMLP. Data represent means ± SE of five independent experiments, 837
with 10 BPHs per replicate. Different letters above the bars indicate significant 838
differences, as determined by a Tukey honest significant difference test (P < 0.05). 839
E, Survival rates of BPH insects feeding on NlMLP-dsRNA-transgenic plants (SG33-2) 840
and WT plants. The experiment was repeated five times, with 20 BPHs per replicate. 841
Data represent means ± SD of five repeats. Asterisks above the columns indicate 842
significant differences compared with WT plants (*, P < 0.05; **, P < 0.01, Student's 843
t-test). 844
F, BPH weight after feeding for 10 days on NlMLP-dsRNA-transgenic plants (SG33-2) 845
and WT plants. The experiment was repeated five times, with 10 BPHs per replicate. 846
Data represent means ± SE of five repeats. Asterisks above the columns indicate 847
significant differences compared with WT plants (*, P < 0.05, Student’s t-test). 848
849
Figure 3. Effects of NlMLP silencing on salivary sheath formation. 850
A and B, Distribution of branch number (A) and length (B) of salivary sheaths formed 851
by BPHs on an artificial diet. BPH insects were fed with dietary sucrose for 48 h and the 852
salivary sheaths were collected and counted under a fluorescence microscope. C, 853
non-injected BPHs; dsGFP, BPHs injected with GFP-dsRNA; dsMLP, BPHs injected 854
with NlMLP-dsRNA. Data represent means ± SE of three repeats. Different letters 855
above the bars indicate significant differences, as determined by a Tukey honest 856
significant difference test (P < 0.05). 857
C, D and E, Scanning electron micrographs showing the morphology of salivary 858
sheaths formed by BPHs on an artificial diet. Salivary sheaths from non-injected BPHs 859
(C) show a complete structure. BPHs injected with GFP-dsRNA (D) formed similar 860
types of sheaths. BPHs injected with NlMLP-dsRNA produced abnormal salivary 861
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33
sheaths (E). Scale bar = 20 μm. 862
F, G and H, Fluorescence microscopy images showing the morphology of salivary 863
sheaths in rice plants. Rice plants were infested with BPH insects for 48 h and 864
investigated by paraffin sectioning. Sheaths produced by: F, non-injected BPHs; G, 865
BPHs injected with GFP-dsRNA; H, BPHs injected with NlMLP-dsRNA. Scale bar = 866
50 μm. 867
868
Figure 4. NlMLP causes cell death in rice protoplasts and N. benthamiana leaves. 869
A, Images of fluorescein diacetate (FDA)-stained viable rice protoplasts transformed 870
with GFP or NlMLP. Living cells were visualized using confocal laser-scanning 871
microscopy and images were taken 20 h after transformation. GFP is a control protein 872
that does not induce cell death. Scale bar = 25 μm. 873
B, Number of viable rice protoplasts transformed with GFP or NlMLP determined after 874
FDA staining. Average values and SEs were calculated from three independent 875
experiments, and 10 randomly selected microscopy fields were counted per experiment. 876
Asterisks above the columns indicate significant differences compared with GFP (**, 877
P < 0.01, Student’s t-test). 878
C, Luciferase (LUC) activity in rice protoplasts co-expressing LUC and NlMLP. Data 879
represent means ± SE of three independent experiments. Asterisks above the columns 880
indicate significant differences compared with GFP (**, P < 0.01, Student’s t-test). 881
D and E, Leaves of N. benthamiana were infiltrated with Agrobacterium tumefaciens 882
carrying GFP, INF1, and NlMLP. The leaves were photographed 5 days after 883
agroinfiltration (D), and the treated leaves were stained with trypan blue (E). INF1, a 884
control protein that induces cell death. GFP, INF1, and NlMLP with the YFP-HA 885
epitope tag; NlMLP-100, NlMLP with no epitope tag. 886
F, Quantification of cell death by measuring electrolyte leakage in N. benthamiana 887
leaves. Electrolyte leakage from the infiltrated leaf discs was measured as a percentage 888
of leakage from boiled discs 4 days after agroinfiltration. Data represent means ± SE of 889
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34
four repeats. Asterisks above the columns indicate significant differences compared 890
with GFP (**, P < 0.01, Student’s t-test). 891
G and H, Silencing of NbMEK2 in N. benthamiana leaves compromises 892
NlMLP-induced cell death but not INF1-induced cell death. GFP, INF1, and NlMLP 893
were transiently expressed in N. benthamiana leaves expressing PVX vector (G) and 894
leaves in which NbMEK2 had been silenced by VIGS (H). The leaves were 895
photographed 5 days after agroinfiltration. 896
I, NbMEK2 transcript abundance in silenced N. benthamiana leaves, as measured by 897
qRT-PCR. Data represent means ± SE of three repeats. Asterisks above the columns 898
indicate significant differences compared with GFP (**, P < 0.01, Student’s t-test). 899
900
Figure 5. NlMLP affects plant defense responses during transfection. 901
A, Aniline blue staining of N. benthamiana leaves showing callose deposition spots in 902
areas transfected with GFP or NlMLP. Photographs were taken 48 h after inoculation. 903
Numbers indicate means ± SE of the number of callose spots in four individual leaf 904
discs. Scale bar = 100 mm. 905
B, Expression of the defense-related genes NbPR3, NbPR4, and NbPR1 following 906
transient expression of GFP and NlMLP in N. benthamiana leaves. Data represent 907
means ± SE of three repeats. Asterisks above the columns indicate significant 908
differences compared with GFP (**, P < 0.01, Student’s t-test). 909
910
Figure 6. Deletion analysis of NlMLP. 911
Left column, schematic diagram of NlMLP and the deletion mutants. 912
Middle column, Relative expression of NbPR4 in N. benthamiana leaves 913
agroinfiltrated with GFP or NlMLP deletion mutants, as determined by qRT-PCR. Data 914
represent means ± SE of three repeats. M, NlMLP or the deletion mutants. Asterisks 915
above the columns indicate significant differences compared with GFP (**, P < 0.01, 916
Student’s t-test). 917
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35
Right column, Cell-death lesions in N. benthamiana leaves expressing the NlMLP 918
deletion mutants. +, +–, and – indicate obvious cell death, weak cell death, and no cell 919
death, respectively. Each experiment was repeated at least three times with similar 920
results. 921
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36
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Figure 1. Molecular characterization of NlMLP.
A, Amino acid sequence of NlMLP. The solid underline indicates the signal peptide
predicted by SignalP. The asterisk (*) indicates the stop codon. The shaded amino acid
residues indicate the peptides detected in BPH-infested rice tissue by LC-MS/MS
analyses. Ser is shown in red. The repeat region is boxed.
B and C, Expression patterns of NlMLP at different developmental stages (B) and in
different tissues (C). Relative levels of NlMLP expression at the indicated
developmental stages (1st to 5
th, 1
st to 5
th instar; F, female adult; M, male adult) and in
different tissues (salivary gland, gut, fat body, and remaining carcass) normalized
against β-actin gene expression, as determined by qRT-PCR. Data represent means ±
SE of three repeats. N = 30. Different letters above the bars indicate significant
differences, as determined by a Tukey honest significant difference test (P < 0.05).
D, mRNA in situ hybridization of NlMLP in salivary glands. Signals from
anti-DIG-FITC (shown in green) bound to a DIG-labeled antisense riboprobe used for
hybridization to NIMLP transcripts were detected by confocal laser-scanning
microscopy. PG, principal gland. AG, accessory gland. Scale bar = 100 μm.
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E, NlMLP localizes to the cytoplasm of rice cells. GFP or NlMLP-GFP fusion protein
was expressed in rice protoplasts by PEG-mediated transformation. Confocal
laser-scanning microscopy was used to investigate fusion protein distribution 16 h
after transformation. Scale bar = 5 μm.
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Figure 2. Effects of NlMLP silencing on BPH feeding and performance.
A, The survival rates of BPH insects after injection were monitored daily. The survival
rates of BPHs injected with dsMLP was significantly reduced compared to BPHs with
no injection and those injected with dsGFP after 2 days of treatment (Student’s t-test).
C, BPHs with no injection. dsGFP, BPHs injected with GFP-dsRNA. dsMLP, BPHs
injected with NlMLP-dsRNA. The experiment was repeated five times, with 10 BPHs
per replicate. Data represent means ± SD of five repeats.
B, Honeydew excretion by BPH insects on filter paper. The intensity of the honeydew
color and area of the honeydew correspond to BPH feeding activity. The experiment
was repeated three times, with 10 filter papers per replicate. Data represent means ± SE
of three repeats. Different letters above the bars indicate significant differences, as
determined by a Tukey honest significant difference test (P < 0.05).
C and D, BPH weight gain (C) and BPH weight gain ratio (D) of BPHs after injection
with dsGFP or dsMLP. Data represent means ± SE of five independent experiments,
with 10 BPHs per replicate. Different letters above the bars indicate significant
differences, as determined by a Tukey honest significant difference test (P < 0.05).
E, Survival rates of BPH insects feeding on NlMLP-dsRNA-transgenic plants (SG33-2)
and WT plants. The experiment was repeated five times, with 20 BPHs per replicate.
Data represent means ± SD of five repeats. Asterisks above the columns indicate
significant differences compared with WT plants (*, P < 0.05; **, P < 0.01, Student's
t-test).
F, BPH weight after feeding for 10 days on NlMLP-dsRNA-transgenic plants (SG33-2)
and WT plants. The experiment was repeated five times, with 10 BPHs per replicate.
Data represent means ± SE of five repeats. Asterisks above the columns indicate
significant differences compared with WT plants (*, P < 0.05, Student’s t-test).
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Figure 3. Effects of NlMLP silencing on salivary sheath formation.
A and B, Distribution of branch number (A) and length (B) of salivary sheaths formed
by BPHs on an artificial diet. BPH insects were fed with dietary sucrose for 48 h and the
salivary sheaths were collected and counted under a fluorescence microscope. C,
non-injected BPHs; dsGFP, BPHs injected with GFP-dsRNA; dsMLP, BPHs injected
with NlMLP-dsRNA. Data represent means ± SE of three repeats. Different letters
above the bars indicate significant differences, as determined by a Tukey honest
significant difference test (P < 0.05).
C, D and E, Scanning electron micrographs showing the morphology of salivary
sheaths formed by BPHs on an artificial diet. Salivary sheaths from non-injected BPHs
(C) show a complete structure. BPHs injected with GFP-dsRNA (D) formed similar
types of sheaths. BPHs injected with NlMLP-dsRNA produced abnormal salivary
sheaths (E). Scale bar = 20 μm.
F, G and H, Fluorescence microscopy images showing the morphology of salivary
sheaths in rice plants. Rice plants were infested with BPH insects for 48 h and
investigated by paraffin sectioning. Sheaths produced by: F, non-injected BPHs; G,
BPHs injected with GFP-dsRNA; H, BPHs injected with NlMLP-dsRNA. Scale bar =
50 μm.
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Figure 4. NlMLP causes cell death in rice protoplasts and N. benthamiana leaves.
A, Images of fluorescein diacetate (FDA)-stained viable rice protoplasts transformed
with GFP or NlMLP. Living cells were visualized using confocal laser-scanning
microscopy and images were taken 20 h after transformation. GFP is a control protein
that does not induce cell death. Scale bar = 25 μm.
B, Number of viable rice protoplasts transformed with GFP or NlMLP determined after
FDA staining. Average values and SEs were calculated from three independent
experiments, and 10 randomly selected microscopy fields were counted per experiment.
Asterisks above the columns indicate significant differences compared with GFP (**,
P < 0.01, Student’s t-test).
C, Luciferase (LUC) activity in rice protoplasts co-expressing LUC and NlMLP. Data
represent means ± SE of three independent experiments. Asterisks above the columns
indicate significant differences compared with GFP (**, P < 0.01, Student’s t-test).
D and E, Leaves of N. benthamiana were infiltrated with Agrobacterium tumefaciens
carrying GFP, INF1, and NlMLP. The leaves were photographed 5 days after
agroinfiltration (D), and the treated leaves were stained with Trypan blue (E). INF1, a
control protein that induces cell death. GFP, INF1, and NlMLP with the YFP-HA
epitope tag; NlMLP-100, NlMLP with no epitope tag.
F, Quantification of cell death by measuring electrolyte leakage in N. benthamiana
leaves. Electrolyte leakage from the infiltrated leaf discs was measured as a percentage
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of leakage from boiled discs 4 days after agroinfiltration. Data represent means ± SE of
four repeats. Asterisks above the columns indicate significant differences compared
with GFP (**, P < 0.01, Student’s t-test).
G and H, Silencing of NbMEK2 in N. benthamiana leaves compromises
NlMLP-induced cell death but not INF1-induced cell death. GFP, INF1, and NlMLP
were transiently expressed in N. benthamiana leaves expressing PVX vector (G) and
leaves in which NbMEK2 had been silenced by VIGS (H). The leaves were
photographed 5 days after agroinfiltration.
I, NbMEK2 transcript abundance in silenced N. benthamiana leaves, as measured by
qRT-PCR. Data represent means ± SE of three repeats. Asterisks above the columns
indicate significant differences compared with GFP (**, P < 0.01, Student’s t-test).
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Figure 5. NlMLP affects plant defense responses during transfection.
A, Aniline blue staining of N. benthamiana leaves showing callose deposition spots in
areas transfected with GFP or NlMLP. Photographs were taken 48 h after inoculation.
Numbers indicate means ± SE of the number of callose spots in four individual leaf
discs. Scale bar = 100 mm.
B, Expression of the defense-related genes NbPR3, NbPR4, and NbPR1 following
transient expression of GFP and NlMLP in N. benthamiana leaves. Data represent
means ± SE of three repeats. Asterisks above the columns indicate significant
differences compared with GFP (**, P < 0.01, Student’s t-test).
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Figure 6. Deletion analysis of NlMLP.
Left column, schematic diagram of NlMLP and the deletion mutants.
Middle column, Relative expression of NbPR4 in N. benthamiana leaves
agroinfiltrated with GFP or NlMLP deletion mutants, as determined by qRT-PCR. Data
represent means ± SE of three repeats. M, NlMLP or the deletion mutants. Asterisks
above the columns indicate significant differences compared with GFP (**, P < 0.01,
Student’s t-test).
Right column, Cell-death lesions in N. benthamiana leaves expressing the NlMLP
deletion mutants. +, +–, and – indicate obvious cell death, weak cell death, and no cell
death, respectively. Each experiment was repeated at least three times with similar
results.
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