Abstract
Salivary elicitors secreted by herbivorous insects can be perceived by
host plants to trigger plant immunity. However, how insects secrete
other salivary components to subsequently attenuate the
elicitor-induced plant immunity remains poorly understood. Here, we
study the small brown planthopper, Laodelphax striatellus salivary
sheath protein LsSP1. Using Y2H, BiFC and LUC assays, we show that
LsSP1 is secreted into host plants and binds to salivary sheath via
mucin-like protein (LsMLP). Rice plants pre-infested with
dsLsSP1-treated L. striatellus are less attractive to L. striatellus
nymphs than those pre-infected with dsGFP-treated controls. Transgenic
rice plants with LsSP1 overexpression rescue the insect feeding defects
caused by a deficiency of LsSP1 secretion, consistent with the
potential role of LsSP1 in manipulating plant defenses. Our results
illustrate the importance of salivary sheath proteins in mediating the
interactions between plants and herbivorous insects.
Subject terms: Effectors in plant pathology, Entomology
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Salivary elicitors secreted by herbivorous insects can be perceived by
host plants to trigger plant immunity. Here, the authors show that the
small brown planthopper salivary sheath protein LsSP1 binds to salivary
sheath proteins and contributes to insect feeding by manipulating rice
plant defenses.
Introduction
In nature, plants are continuously challenged by various pathogens,
including bacteria, fungi, and nematodes. To survive or fend off
attacks, plants have evolved multi-layered immune systems from
recognizing pathogens to activating defense responses. Pattern
recognition receptors can perceive “non-self” molecules and activate
the pattern-triggered immunity (PTI), including mitogen-activated
protein kinase (MAPK) cascades, reactive oxygen species (ROS), and
hormone signaling^[54]1–[55]3. To counteract plant immunity, plant
pathogens deliver secretory effectors to target the immune signaling
components of PTI and interfere with their activities^[56]4,[57]5.
However, some effectors can be sensed by the plants with time, further
initiating the effector-triggered immunity^[58]6. Over millions of
years of co-evolution, plant pathogens have developed dynamic and
complex interactions with host plants.
Piercing-sucking insects, such as planthoppers, aphids, and whiteflies,
are important pests that damage host plants by feeding or transmitting
viruses. During the feeding process, two types of saliva (gel saliva
and watery saliva) are ejected into plant tissues^[59]7. These oral
secretions, on the one hand, hinder insect performance by activating
plant defenses. For example, salivary protein Cathepsin B3 from Myzus
persicae can be recognized by Nicotiana tabacum plants, which thus
suppress aphid feeding by triggering ROS accumulation^[60]8. Moreover,
salivary protein 1 from Nilaparvata lugens induces cell death, H[2]O[2]
accumulation, defense-related gene expression, and callose deposition
when it is transiently expressed in Nicotiana benthamiana leaves or
rice protoplasts^[61]9. On the other hand, saliva exerts multiple roles
in improving insect performance, such as calcium binding proteins for
calcium regulation^[62]10, DNase II for extracellular DNA
degradation^[63]11, and Helicoverpa armigera R-like protein 1 (HARP1)
for plant hormonal manipulation^[64]12. Piercing-sucking herbivores
eject abundant salivary effectors into plant tissues. There may be some
salivary elicitors triggering plant defenses, while the
elicitor-induced plant defenses are inhibited by other salivary
components. However, little is known about the complex interactions in
saliva.
Formed by gel saliva, salivary sheath is indispensable for insect
feeding. It is secreted during the stylet probing, which provides
mechanical stability and lubrication for stylet movement^[65]13. The
salivary sheath is capable of sealing the stylet penetration site,
thereby preventing the plant immunity triggered by leaked cell
components^[66]7. In aphids and planthoppers, the disrupted salivary
sheath formation can hinder insect feeding from plant sieve tubes, but
not from the artificial diet^[67]14,[68]15. After secretion, salivary
sheath is distributed in plant apoplast and directly contacts with
plant cells^[69]15. Salivary sheath is composed by many salivary sheath
proteins, which can be potentially recognized as herbivore-associated
molecular patterns (HAMPs) that activate the immune response in host
plants^[70]16. Because of the forward roles in herbivore–plant
interactions, a few proteins in the salivary sheath may exhibit a high
evolutionary rate^[71]17. Nevertheless, the current knowledge on
salivary sheath is mainly limited on its mechanical function.
Therefore, it is interesting to reveal its other functions in
herbivore–plant interactions.
Plant apoplast space is an important battleground between the host and
pathogens^[72]18. The papain-like cysteine proteases (PLCPs), which
share a conserved protease domain, are prominent enzymes in the plant
apoplast that can function as the central hubs in plant
immunity^[73]19. As a well-known maize insect resistance gene, Mir1
belongs to PLCPs^[74]20. It can be rapidly accumulated at the wound
sites, and can degrade the insect gut surface to confer maize
resistance against caterpillars^[75]20,[76]21. Mir1 accumulation is
reported to enhance plant resistance against root-feeding herbivores
and corn leaf aphids^[77]22,[78]23. In turn, PLCPs are the common
targets of pathogen effectors. Fungi, oomycete, nematodes, and bacteria
can actively interfere with the activity or subcellular location of
plant PLCPs, which can thereby suppress plant immunity^[79]24–[80]28.
The small brown planthopper, Laodelphax striatellus, is a destructive
pest that causes severe yield reductions and economic losses in rice
crops. Similar to most phloem-feeding insects, planthoppers can secrete
a mixture of saliva during feeding. Several salivary proteins have been
found to participate in salivary sheath formation and/or interfere with
the host immune responses^[81]13,[82]29. Nevertheless, the functions of
most salivary proteins remain unknown. In this study, L. striatellus
salivary sheath protein LsSP1 is employed as a molecular probe to
investigate the mechanism by which this planthopper can interact with
salivary sheath mucin-like protein (LsMLP)-triggered, PLCP-mediated
plant defenses. The salivary LsSP1 is secreted into host plants during
feeding and is shown to interact with multiple PLCPs belonging to
different subfamilies in yeast two hybrid (Y2H) and bimolecular
fluorescence complementation (BiFC) assays. OsOryzain is a member of
PLCPs. Expression of LsSP1 in N. benthamiana plants significantly
attenuates the H[2]O[2] accumulation and defense gene expression
induced by OsOryzain and LsMLP, while in rice plants the role of
OsOryzain is not confirmed. Overexpression of LsSP1 in rice plants
rescues the feeding defects caused by a deficiency in LsSP1 secretion.
Results
LsSP1 is important for L. striatellus feeding on rice plants
Many genes that highly expressed in L. striatellus salivary glands were
planthopper-specific^[83]30, and their homologous genes were not found
in other species (Supplementary Data [84]1). To reveal their specific
roles in the planthopper-rice interactions, this study firstly
investigated the expression patterns of these genes in different
tissues. In total, 30 genes were found to be specifically expressed in
salivary glands (Supplementary Fig. [85]1). The L. striatellus salivary
protein 1 (hereafter: LsSP1, accession number: [86]ON322955) was among
the top 5 most abundant, salivary gland-specific, and
planthopper-specific genes, which was therefore selected for further
analysis. The insect survivorship was not significantly affected by
treating L. striatellus with dsLsSP1 (log-rank test, p = 0.3044;
Fig. [87]1a). However, the dsLsSP1-treated L. striatellus produced less
offspring (one-way ANOVA test followed by Tukey’s multiple comparisons
test, p = 0.0153; Fig. [88]1b) and excreted less honeydew (one-way
ANOVA test followed by Tukey’s multiple comparisons test, p = 0.0127;
Fig. [89]1c) than the dsGFP-treated control. Electrical penetration
graph (EPG) was adopted for monitoring the insect feeding behavior.
Compared with dsGFP treatment, L. striatellus treated with dsLsSP1
exhibited a significant decrease (by 62%; two-tailed unpaired Student’s
t test, p = 0.0057) in phloem sap ingestion, along with the slight
increases in nonpenetration (by 23%; two-tailed unpaired Student’s t
test, p = 0.2769) and pathway duration phase (by 31%; two-tailed
unpaired Student’s t test, p = 0.2525) (Fig. [90]1d, e). These results
indicate that LsSP1 plays a role in L. striatellus feeding on rice
plants.
Fig. 1. Effect of dsRNA treatment on Laodelphax striatellus.
[91]Fig. 1
[92]Open in a new tab
a–c Effect of LsSP1 knockdown on L. striatellus survival rate (a),
fecundity (b), and honeydew excretion (c). The untreated (CK) and
dsGFP-treated L. striatellus were used as controls. Data in a are
presented as mean values ±95% confidence intervals (displayed in light
shades). Different lowercase letters indicate statistically significant
differences at P < 0.05 level according to log-rank test (a) or one-way
ANOVA test followed by Tukey’s multiple comparisons test (b, c). d
Comparison of electrical penetration graph (EPG) parameters between
dsGFP-treated and dsLsSP1-treated L. striatellus. All EPG recordings
were performed for 8 h. P-values were determined by two-tailed unpaired
Student’s t test. **P < 0.01; ns, not significant. Data in b–d are
presented as mean values ±SEM. For survival analysis in a, n = 91,
n = 88, and n = 106 individuals in CK, dsGFP, and dsLsSP1,
respectively; for fecundity analysis in b, n = 20 independent
biological replicates in three treatments; for honeydew analysis in c,
n = 16, n = 25, and n = 23 independent biological replicates in CK,
dsGFP, and dsLsSP1, respectively; for EPG analysis in d, n = 11
independent biological replicates in three treatments. e Overall
typical EPG waveforms over 1 h for dsGFP-treated (upper) and
dsLsSP1-treated L. striatellus (lower). The insect feeding behavior was
classified into nonpenetration (np), pathway duration (N1 + N2 + N3),
phloem sap ingestion (N4), and xylem sap ingestion (N5) phases. The
rice variety cv. ASD7 was used. Source data are provided as a Source
Data file.
LsSP1 is a salivary sheath protein not essential for salivary sheath
formation
LsSP1 contained an open reading frame of 771 bp, encoding a protein of
256 amino acids. No conserved domain was found in LsSP1. The protein
possessed an N-terminal signal peptide, with no transmembrane domain,
which indicated its secretory property (Supplementary Fig. [93]2a).
Homologous analysis demonstrated that LsSP1 was a planthopper-specific
protein, and exhibited 43.9% and 58.5% amino acid sequence identities
to secretory proteins in the brown planthopper N. lugens ([94]ASL05017)
and the white-backed planthopper Sogatella furcifera ([95]ON322954),
respectively (Supplementary Fig. [96]2b). LsSP1 and its homologous
genes in other planthopper species have not been well investigated
previously. Spatial-temporal expression analysis showed that LsSP1 was
mainly expressed at the nymph and adult stages (Supplementary
Fig. [97]2c), and immunohistochemical (IHC) staining showed that LsSP1
was exclusively expressed in a pair of follicles in primary salivary
glands (Fig. [98]2a, b). The transcript level of LsSP1 was reduced by
90% after the treatment of L. striatellus with dsLsSP1, and almost no
LsSP1 signal was detected in salivary glands (Supplementary
Fig. [99]3a–c). LsSP1 was secreted during insect feeding, and a band of
approximately 35 kDa was detected in rice plants infested by L.
striatellus, but not in the non-infested plants (Fig. [100]2c).
Fig. 2. LsSP1 is a salivary sheath protein and secreted into plants.
[101]Fig. 2
[102]Open in a new tab
a Immunohistochemical staining of LsSP1 in salivary glands. L.
striatellus salivary glands were incubated with anti-LsSP1 serum
conjugated with Alexa Fluor™ 488 NHS Ester (green) and actin dye
phalloidinrhodamine (red) and examined by Leica SP8. The nucleus was
stained with DAPI (blue). The lower images represent the enlarged
images of the boxed area in the upper image. The boxed area was
indicated in bright filed image. b Expression patterns of LsSP1 in
different tissues quantified by qRT-PCR (upper) and western-blotting
(lower) assays. FB fat body, SG salivary gland, Ca carcass, Te testis,
Ov ovary. Data are presented as mean values ± SEM (n = 3 independent
biological replicates). Different lowercase letters indicate
statistically significant differences at P < 0.05 level according to
one-way ANOVA test followed by Tukey’s multiple comparisons test. c
Detection of LsSP1 in untreated plants (lane 1) and plants infested by
L. striatellus (lane 2), watery (lane 3) and gel saliva (lane 4). d, e
LsSP1 staining of salivary sheath on parafilm (d) and in rice tissues
(e). Green, LsSP1; blue, nucleus. Coomassie brilliant blue (CBB)
staining was conducted to visualize the amount of sample loading.
Experiments in a, c, d, and e were repeated three times with the
similar results. Source data are provided as a Source Data file.
For most of the piercing-sucking insects, two types of saliva (gel and
watery saliva) are ejected into plant tissues during the feeding
process. Previously, the components of L. striatellus watery saliva
collected by artificial diet were reported^[103]30. However, LsSP1 was
not detected in those samples. Thereafter, the salivary sheath (gel
saliva) was collected from the inner layer of the Parafilm membrane to
investigate whether LsSP1 existed in the salivary sheath. As a result,
a band of LsSP1 was detected in the salivary sheath sample
(Fig. [104]2c). By contrast, the band of LsSP1 in watery saliva sample
was not visible, indicating that LsSP1 was a salivary sheath protein.
Immunohistochemistry (IHC) staining analysis of salivary sheath on the
Parafilm membrane and in rice plants further confirmed the presence of
LsSP1 in salivary sheath (Fig. [105]2d, e), whereas almost no signal
was detected in salivary sheath secreted from dsLsSP1-treated L.
striatellus (Supplementary Fig. [106]3d, e). LsSP1 deficiency did not
influence salivary sheath formation, and there was no significant
difference in salivary sheath appearance between dsLsSP1 treatment and
the control as observed under scanning electron microscopy (SEM;
Supplementary Fig. [107]4). Also, we did not find significant
difference in the length of salivary sheath on the Parafilm membrane
(two-tailed unpaired Student’s t test, p = 0.5926; measured from the
top to base of salivary sheath under SEM) or the number of salivary
sheaths left on the rice surface (two-tailed unpaired Student’s t test,
p = 0.7615; measured by counting the ring-shaped salivary sheath
structure under SEM) after dsLsSP1 treatment (Supplementary
Fig. [108]4). These results suggest that LsSP1 is a salivary sheath
protein, but that it is not indispensable for salivary sheath
formation, which is significantly different from two previously
reported salivary sheath proteins^[109]14,[110]31.
LsSP1 binds to the salivary sheath protein mucin-like protein LsMLP using
Y2H, BiFC and LUC assays
Our previous work demonstrated that mucin-like protein (MLP) was the
main component of salivary sheath in the planthopper N. lugens^[111]31.
Amino acid alignment demonstrated that MLPs among three planthoppers
were highly homologous (Supplementary Fig. [112]5a). At first, the
function of L. striatellus MLP (LsMLP, accession number: [113]ON568348)
was investigated by RNAi (Supplementary Fig. [114]5b). The
LsMLP-deficient L. striatellus only secreted the short salivary sheath
(two-tailed unpaired Student’s t test, p < 0.001; Supplementary
Fig. [115]6), similar to that of NlMLP-deficient N. lugens^[116]16. The
number of salivary sheaths left on the rice plant significantly
decreased when L. striatellus was treated with dsLsMLP (two-tailed
unpaired Student’s t test, p = 0.015; Supplementary Fig. [117]6).
Furthermore, the LsMLP-deficient L. striatellus exhibited a high
mortality rate (log-rank test, p < 0.001; Supplementary Fig. [118]5c),
indicating that LsMLP is important for L. striatellus performance.
Meanwhile, the treatment of L. striatellus with dsLsMLP did not
influence LsSP1 at the transcript (two-tailed unpaired Student’s t
test, p = 0.5317; Fig. [119]3a) or the protein level (Fig. [120]3b).
However, almost no fluorescence signal of LsSP1 was detected in
salivary sheath secreted from dsLsMLP-treated L. striatellus, which was
significantly different from that of dsGFP-treated control
(Fig. [121]3c). Thereafter, this study detected whether LsSP1 existed
in watery saliva or salivary sheath secreted from dsLsMLP-treated L.
striatellus. Interestingly, more LsSP1 was found in watery saliva than
in salivary sheath collected from dsLsMLP-treated L. striatellus,
perhaps indicating that LsSP1 fails to bind to salivary sheath in the
absence of LsMLP (Fig. [122]3d).
Fig. 3. LsSP1 binds to a salivary sheath protein LsMLP.
[123]Fig. 3
[124]Open in a new tab
a, b Influence of dsLsMLP treatment on the LsSP1 abundance in salivary
gland quantified by qRT-PCR (a) and western-blotting (b) assays. Data
are presented as mean values ±SEM (n = 3 independent biological
replicates). P-values were determined by two-tailed unpaired Student’s
t test. ns, not significant. c LsSP1 staining of salivary sheath
secreted from dsRNA-treated Laodelphax striatellus. The salivary
sheaths secreted from dsGFP- or dsLsMLP-treated L. striatellus were
probed with the anti-LsSP1 serum conjugated with Alexa Fluor™ 488 NHS
Ester (green), and examined by Leica SP8. Bars = 20 μm. d Detection of
LsSP1 in watery (lane1, lane3) and gel saliva (lane 2, lane 4) secreted
from dsGFP- or dsLsMLP-treated L. striatellus. e Yeast two hybrid
assays showing the interaction between LsSP1 and LsMLP. The different
combinations of constructs transformed into yeast cells were grown on
the selective medium SD/-Trp/-Leu (DDO), and the interactions were
tested with SD/-Trp/-Leu/-His/-Ade (QDO). f Bimolecular fluorescence
complementation (BiFC) assays showing the interaction between LsSP1 and
LsMLP. Bars = 20 μm. g The co-expression scheme in Nicotiana
benthamiana leaves during Luciferase complementation (LUC) assays. h
Results from LUC assays showing the interaction between LsSP1 and
LsMLP. Bar = 1 cm. Experiments in b, c, d, f, and h were repeated three
times with the similar results. Source data are provided as a Source
Data file.
The potential interaction between LsSP1 and LsMLP was investigated
using point-to-point Y2H assays. The yeast transformants expressing
DNA-binding domain (BD)-LsMLP and activating domain (AD)-LsSP1 were
found to grow on the quadruple dropout medium, which was not observed
in transformants bearing the control constructs (Fig. [125]3e). Similar
results were found in yeast transformants expressing BD-LsSP1 and
AD-LsMLP (Fig. [126]3e). Also, the interaction between LsSP1 and LsMLP
was verified by BiFC assay (Fig. [127]3f) and luciferase
complementation (LUC) assay (Fig. [128]3g, h). These results may
suggest that LsSP1 interacts with LsMLP in vivo.
LsSP1 can interact with rice papain-like cysteine proteases using Y2H,
GST-pull down, BiFC, and LUC assays
To understand the potential roles of LsSP1 in insect-plant interaction,
Y2H screening was performed using a rice cDNA library. Seven proteins
were found to potentially interact with LsSP1, including an Oryza
sativa Oryzain (OsOryzain, [129]NP_001389372.1, LOC_Os04g55650)
(Supplementary Table [130]1). OsOryzain was highly homologous with
Arabidopsis RD21, tomato C14, and maize Mir3 cysteine proteases. It
contained a predicted N-terminal secretion signal and a self-inhibitory
prodomain followed by the peptidase and granulin domains (Supplementary
Fig. [131]7a). OsOryzain is a member of PLCPs that act as a central hub
in plant immunity and are required for the full resistance of plants to
various pathogens^[132]19. In tomato, C14 is converted into immature
(iC14) and mature (mC14) isoforms that are accumulated into various
subcellular compartments and the apoplast^[133]28. The interaction
between OsOryzain and LsSP1 was confirmed using point-to-point Y2H,
GST-pull down, BiFC, and LUC assays (Supplementary Note 1 and
Supplementary Fig. [134]7b–f). Furthermore, our experiments show that
LsSP1 is capable of interacting with multiple PLCPs belonging to
different subfamilies using point-to-point Y2H and BiFC assays
(Supplementary Note [135]1 and Supplementary Figs. [136]8 and [137]9).
Induction of PLCPs by L. striatellus infestation and salicylic acid (SA)
treatment using rice plants
Relative transcript levels of PLCPs in response to L. striatellus
infestation were investigated. Among the 46 PLCPs investigated, 8 were
found to be significantly induced upon L. striatellus infestation
(Supplementary Fig. [138]10). The expression of OsOryzain was induced
at 3 h post-infestation, and reached a peak at 6 h (Supplementary
Fig. [139]11a). Salicylic acid (SA) exerts a critical role in plant
defense against sap-sucking herbivores^[140]32–[141]34. The induction
of SA biosynthetic genes and SA responsive genes was detected upon L.
striatellus infestation (Supplementary Fig. [142]12). To investigate
the possible role of SA in regulating PLCPs, the relative transcript
levels of PLCPs were quantified after SA treatment. As a result, SA
significantly induced the expression of 7 PLCPs, including OsOryzain
(Supplementary Figs. [143]10 and [144]11a). These results indicate that
numerous PLCPs might be associated with the L. striatellus-induced
SA-mediated plant defenses in rice plants.
In addition, our experiments also investigated the protein levels of
OsOryzain in response to SA treatment and L. striatellus infestation.
The results demonstrated that SA treatment and L. striatellus
infestation induced the expression of OsOryzain in plant cells, while
rice plants infested by L. striatellus secreted a lower amount of
mature OsOryzain (mOsOryzain) into apoplast than that under SA
treatment (Supplementary Note [145]2 and Supplementary Fig. [146]11b,
c). We were not able to confirm that OsOryzain is involved in the plant
defense response to L. striatellus, hence additional methods and
results involving OsOryzain experiments have been moved to the
Supplementary Note [147]3–[148]4, Supplementary Methods, and
Supplementary Figs. [149]13–[150]15.
LsSP1 affects plant defenses in rice plants
To determine whether LsSP1 affects plant defenses in rice plants, the
feeding preference of L. striatellus nymphs on plants pre-infested by
dsGFP- and dsLsSP1-treated L. striatellus was compared. The results
revealed that rice plants pre-infested with dsLsSP1-treated L.
striatellus were less attractive to L. striatellus nymphs than those
pre-infected with dsGFP-treated controls (Fig. [151]4a), suggesting
that dsLsSP1-treated L. striatellus might elicit plant defenses and
become less palatable to conspecifics.
Fig. 4. Influences of dsRNA-treated Laodelphax striatellus on rice plants.
[152]Fig. 4
[153]Open in a new tab
a The attraction of rice plants infested by dsRNA-treated L.
striatellus to nymphs in the two-choice equipment. Data are presented
as mean values ±SEM (n = 19 independent biological replicates).
P-values were determined by two-tailed unpaired Student’s t test.
**P < 0.01; ***P < 0.001; ns, not significant. b Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analysis of differentially
expressed genes (DEGs). Enriched P-values were calculated according to
one-sided hypergeometric test using TBtools software^[154]70. c
Upregulation of salicylic acid (SA)-related genes in dsLsSP1-treated L.
striatellus infested plants compared with dsGFP-treated L. striatellus
infested ones. d H[2]O[2] levels in the untreated rice plants and rice
plants infested by dsRNA-treated L. striatellus. Different lowercase
letters indicate statistically significant differences at P < 0.05
level according to one-way ANOVA test followed by Tukey’s multiple
comparisons test. e Upregulation of defense genes in dsLsSP1-treated L.
striatellus infested plants compared with dsGFP-treated L. striatellus
infested ones. P-values were determined by two-tailed unpaired
Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not
significant. PAD4 phytoalexin deficient 4, SAMT SA methyl transferase,
SAGT SA glucosyl transferase, WRKY transcription factor WRKY, PR1
pathogenesis-related 1. Data in d and e are presented as mean values
±SEM (n = 3 biological replicates). The rice variety cv. ASD7 was used.
Source data are provided as a Source Data file.
Thereafter, plants infested by dsGFP-treated L. striatellus and
dsLsSP1-treated L. striatellus were subject to transcriptomic
sequencing. In total, 405 differentially expressed genes (DEGs) were
identified, among which 90.9% were up-regulated in dsLsSP1-treated L.
striatellus infested plants (Supplementary Fig. [155]16 and
Supplementary Data [156]2). Enrichment analysis demonstrated that the
majority of DEGs were involved in plant-pathogen interaction,
environmental adaptation, transporters, plant hormone signal
transduction, and terpenoids metabolism (Fig. [157]4b). Among the 28 SA
biosynthetic or SA responsive genes (Supplementary Table [158]2), 8
were found to be differentially expressed. These DEGs were all
up-regulated (Fig. [159]4c), indicating the activation of SA pathway in
dsLsSP1-treated L. striatellus infested plants compared with
dsGFP-treated L. striatellus infested ones. H[2]O[2] accumulation has
been applied as a marker for plant basal defenses against sap-sucking
herbivores^[160]29,[161]35. In this study, H[2]O[2] levels in rice
plants were significantly higher at 24 h after dsLsSP1-treated L.
striatellus infestation than those after dsGFP-treated L. striatellus
infestation (one-way ANOVA test followed by Tukey’s multiple
comparisons test, p = 0.0298; Fig. [162]4d). Quantitative real-time PCR
(qRT-PCR) analysis further confirmed the upregulation of defense genes
(Fig. [163]4e), and the obtained results were consistent with
transcriptomic data. Collectively, these results demonstrate that a
deficiency in LsSP1 secretion activates plant defenses as a response to
L. striatellus infestation.
Overexpressing LsSP1 in rice plants benefits dsLsSP1-treated L. striatellus
feeding
Transgenic Nipponbare rice plants with constitutive LsSP1
overexpression were constructed (Supplementary Fig. [164]17). The
wild-type (WT) Nipponbare plant was used as a control. Two independent
homozygous lines were used, and similar results were obtained. The
results of comparison group 1 (WT and oeSP1#1) and comparison group 2
(WT and oeSP1#2) are presented in Fig. [165]5 and Supplementary
Fig. [166]18, respectively. The resistance of transgenic plants to L.
striatellus (4th instar; wild-type) infestation was firstly
investigated. No significant resistance changes in oeSP1 plants were
found when compared with WT plants (two-tailed unpaired Student’s t
test, p = 0.4880 in comparison group 1, p = 0.6704 in comparison group
2; Supplementary Fig. [167]19). Compared with dsGFP-treated controls,
the treatment of L. striatellus with dsLsSP1 did not affect insect
survivorship after feeding on oeSP1 plants (log-rank test, p = 0.9913
on oeSP1#1, p = 0.5715 on oeSP1#2; Supplementary Fig. [168]20). For
fecundity analysis, the dsLsSP1-treated L. striatellus produced less
offspring than the dsGFP-treated control when feeding on WT plants
(two-tailed unpaired Student’s t test, p = 0.0299; Fig. [169]5a).
However, this detrimental effect was not observed when dsLsSP1-treated
L. striatellus fed on oeSP1 plants (two-tailed unpaired Student’s t
test, p = 0.8771 in comparison group 1, p = 0.7670 in comparison group
2; Fig. [170]5a and Supplementary Fig. [171]18a). For honeydew
excretion, the dsLsSP1-treated L. striatellus excreted less honeydew
than the dsGFP-treated control when feeding on WT plants, although with
no statistical significance (two-tailed unpaired Student’s t test,
p = 0.1047; Fig. [172]5b). There was also no significant difference in
honeydew excretion between dsGFP- and dsLsSP1-treated L. striatellus
when feeding on oeSP1 plants (two-tailed unpaired Student’s t test,
p = 0.3751 in comparison group 1, p = 0.9523 in comparison group 2;
Fig. [173]5b and Supplementary Fig. [174]18b). EPG was subsequently
used to monitor the insect feeding behavior on transgenic plants.
Compared with dsGFP-treated controls, the dsLsSP1-treated L.
striatellus exhibited a significant decrease in phloem sap ingestion
when feeding on WT plants (two-tailed unpaired Student’s t test,
p = 0.0426 in comparison group 1, p = 0.0037 in comparison group 2;
Fig. [175]5c and Supplementary Fig. [176]18c). Nevertheless, no
significant difference in phloem sap ingestion was observed between
dsGFP- and dsLsSP1-treated L. striatellus feeding on oeSP1 plants
(two-tailed unpaired Student’s t test, p = 0.8913 in comparison group
1, p = 0.9390 in comparison group 2; Fig. [177]5c and Supplementary
Fig. [178]18c), indicating that overexpression of LsSP1 in rice plants
rescued the feeding defects caused by a deficiency in LsSP1 secretion.
Fig. 5. Influences of Laodelphax striatellus infestation on wild type (WT)
and oeSP1#1 plants.
[179]Fig. 5
[180]Open in a new tab
a–c Comparison of fecundity (a), honeydew excretion (b), and electrical
penetration graph (EPG) parameters (c) between dsGFP-treated and
dsLsSP1-treated L. striatellus on WT and oeSP1#1 plants. P-values were
determined by two-tailed unpaired Student’s t test. *P < 0.05;
**P < 0.01; ***P < 0.001; ns, not significant. Data are presented as
mean values ±SEM. For fecundity (a) and honeydew (b) analysis, n = 20
independent biological replicates in each treatment; for EPG analysis
(c), n = 14 (WT-dsGFP), n = 16 (WT-dsLsSP1), n = 13 (oeSP1#1-dsGFP),
and n = 13 (oeSP1#1-dsLsSP1) independent biological replicates. All EPG
recordings were performed for 8 h. N1 + N2 + N3 pathway duration, N4
phloem sap ingestion, N5 xylem sap ingestion, np nonpenetration. d
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis of DEGs specifically identified in WT plants, but not in
oeSP1#1 plants. Enriched P-values were calculated according to
one-sided hypergeometric test using TBtools software^[181]70. e The
impacts of L. striatellus infestation on salicylic acid (SA) marker
genes in WT and oeSP1#1 plants. PAL phenylalanine ammonia lyase, PAD4
phytoalexin deficient 4, GRX480 glutaredoxin GRX480, SAMT SA methyl
transferase, ICS isochorismate synthase, WRKY transcription factor
WRKY, NPR1 non-repressor of pathogenesis-related protein 1, PR1
pathogenesis-related 1, SAGT SA glucosyl transferase. Nipponbare rice
plants and transgenic plants of Nipponbare background were used. Source
data are provided as a Source Data file.
To comprehensively illustrate the effects of LsSP1 on rice plants,
transcriptomic analyses were performed on WT and oeSP1#1 plants that
were untreated or infested by dsLsSP1-treated L. striatellus. DEGs
between untreated and dsLsSP1-treated L. striatellus infested plants
were compared, and totally 3396 and 1998 genes were identified in WT
and oeSP1#1 plants, respectively (Supplementary Data [182]3–[183]4).
There were 2335 DEGs specifically identified in WT plants, but not in
oeSP1#1 plants, and they were potentially correlated with
LsSP1-associated responses. Enrichment analysis revealed that the
majority of these genes were involved in plant hormone signal
transduction, plant-pathogen interaction, MAPK signal transduction, and
amino acid metabolism (Fig. [184]5d). Among the 28 SA-related genes, 18
were differentially expressed in at least one comparison group, and 16
were significantly up-regulated after infestation (Fig. [185]5e).
Interestingly, these up-regulated genes were induced to a lower extent
in oeSP1#1 plants compared with those in WT plants (Fig. [186]5e),
indicating that LsSP1 overexpression attenuated the L.
striatellus-induced SA biosynthesis and SA response.
Discussion
Herbivorous insects have developed dynamic and complex interactions
with host plants. Advanced understanding towards their underlying
mechanisms will provide the fundamental knowledge for developing
efficient pest management strategies. In this study, the role of
salivary LsSP1 in its interaction with rice hosts was investigated.
Using Y2H, BiFC and LUC assays, we showed that LsSP1 was secreted into
plant tissues during feeding and directly interacted with the salivary
sheath protein LsMLP. In yeast and N. benthamiana, LsSP1 interacted
with multiple PLCPs in various subfamilies. LsSP1 knockdown led to a
decrease in insect feeding and reduced insect reproduction on WT
plants, but not on oeSP1 plants. Our results indicate that the salivary
sheath protein LsSP1, although not essential for salivary sheath
formation, is beneficial for insect performance.
During the feeding process, herbivorous insects can secrete hundreds of
proteins into plant tissues. Previously, most salivary proteins are
investigated individually, and it is found that different salivary
proteins from one species exerted diversified roles in insect-plant
interactions^[187]36,[188]37. For example, in M. persicae, the
overexpression of salivary protein Mp10 activates multiple defense
pathways in N. benthamiana plants and reduces aphid
performance^[189]36,[190]38. However, the overexpression of another
salivary protein Mp55 increases the attraction of N. benthamiana plants
to aphids, and promotes aphid performance^[191]37. L. striatellus can
successfully ingest rice phloem saps with limited plant defenses.
However, when LsMLP was overexpressed, elevated accumulation of
H[2]O[2]was detected (Supplementary Fig. [192]15), which was in
contradiction with the actual feeding situation. Therefore, there must
exist other salivary components responsible for attenuating the
LsMLP-induced plant defenses or masking the LsMLP signal. To the best
of our knowledge, no such case has been reported in this insect
species, although several proteins in aphids and mirid bug are found to
be capable of inhibiting the plant defenses triggered by bacterial
flag22 or oomycete INF1^[193]38–[194]40. Our study demonstrated that
LsSP1 bound to LsMLP directly, providing clues that LsSP1 may prevent
the activation of plant defenses by masking the LsMLP, which deserves
further investigation.
Apoplastic PLCPs act in the front line of plant immunity against a wide
range of pathogens, including fungi, bacteria, and oomycetes^[195]41.
Depletion or knockdown of proteases such as Rcr3, RD19, and Pip1
significantly decreases the plant susceptibility to the invading
pathogens^[196]42–[197]44. In maize, PLCPs are required to release the
bioactive Zip1, a small peptide that activates SA signaling^[198]45. In
turn, Zip1 release will enhance PLCP activity, thereby establishing a
positive feedback loop and promoting the SA-mediated defenses^[199]45.
Our study demonstrated that rice genes related to SA signaling were
differentially expressed when infested by L. striatellus, and OsOryzain
was significantly induced upon SA treatment and L. striatellus
infestation (Supplementary Figs. [200]11 and [201]12). This result
might be an indicator that OsOryzain is regulated through SA pathway.
SA signaling plays an important role in the rice defense against
planthoppers^[202]33. The transcript level of OsOryzain reached a peak
at 6 h-post L. striatellus infestation, while a peak was reached at 12
h-post SA treatment (Supplementary Fig. [203]11a). The different
induction patterns indicated that other factors, in addition to SA
pathway, might also be responsible for OsOryzain expression, which
deserves further investigation.
Although our study showed interaction between LsSP1 and OsOryzain in
Y2H assays (Supplementary Note [204]1 and Supplementary Fig. [205]7),
rice plants knockout of OsOryzain cannot well rescue the feeding
defects caused by a deficiency in LsSP1 secretion as that LsSP1
overexpressing plants did (Supplementary Note [206]5, Fig. [207]5, and
Supplementary Fig. [208]21). This might be explained by complex
interactions between effectors and different plant defense actors. As a
case in Phytophthora, the multifunctional effector Avrblb2 can
neutralize host defense proteases via targeting PLCPs^[209]28, and
suppresses defense associated Ca^2+ signaling pathway by interacting
with host calmodulin^[210]46. For salivary LsSP1, it targets multiple
PLCPs belonging to different subfamilies (Supplementary Fig. [211]8).
The knockout of OsOryzain alone cannot inhibit plant defenses initiated
by other PLCPs. In addition, LsSP1 is capable of interacting with other
plant and insect proteins (Fig. [212]3 and Supplementary Table [213]1).
The salivary LsSP1 potentially exerts multiple roles during insect
feeding, and affects plant defense in other ways independent of PLCPs,
which deserves further investigation.
Methods
Insects and plants
The L. striatellus strain was originally collected from a rice field in
Ningbo China. The insects and rice plants were maintained in a climate
chamber at 25 ± 1 °C, with 70–80% relative humidity, and a light/dark
photoperiod of 16/8 h. Two rice varieties (cv. ASD7 and Nipponbare)
were used in this study. The resistant variety ASD7, which contained
the brown planthopper resistance gene BPH2, was also reported to confer
resistance to small brown planthopper^[214]47,[215]48, and was
extensively applied for insect bioassays. As the transgenic rice plants
generated in this study were of Nipponbare background, wild-type
Nipponbare plants were used as a control. Therefore, the rice variety
used in transgenic rice plant analysis was Nipponbare. For the rest of
rice-associated experiments, ASD7 plants were used. In addition, N.
benthamiana plants were kept in a growth chamber at 23 ± 1 °C under a
light/dark photoperiod of 16 h/8 h.
Analysis of genes abundantly expressed in salivary glands
The top 100 genes that were abundantly expressed in L. striatellus
salivary glands were reported in our previous study^[216]30. To
identify the potential planthopper-specific genes, these 100 genes were
first subject to BLAST search against the predicted proteins in
Acyrthosiphon pisum;^[217]49 Bemisia tabaci;^[218]50 Riptortus
pedestris;^[219]51 Homalodisca vitripennis^[220]52, and Drosophila
melanogaster^[221]53, with a cutoff E-value of 10^-5, respectively.
Genes with no homology in the above species were subsequently searched
against the NCBI nr database. Only genes with distributions restricted
to three planthoppers (L. striatellus, S. furcifera, and N. lugens)
were defined as the planthopper-specific genes. Thereafter, the
expression patterns of top 100 genes in different tissues were
investigated based on the transcripts per million (TPM) expression
values. The TPM expression values of L. striatellus genes were
generated by analyzing the transcriptomic data of salivary gland, gut,
fat body, carcass, testis, and ovary, and used in our laboratory to
preliminarily investigate the gene expression patterns. The TPM
expression values of the top 100 genes were displayed in Supplementary
Data [222]1. For the identification of salivary gland-specific genes,
TPM of each gene in salivary gland was compared with that in the other
five tissues, respectively. Afterwards, the gene-relative abundance
(ratio) in each comparison group was calculated. Genes with fold
changes>10 in all comparison groups were considered to be salivary
gland-specific genes.
Sequence analysis
The SignalP 5.0 Server ([223]https://services.healthtech.dtu.dk) was
adopted for predicting the presence of signal peptides and cleavage
sites. Transmembrane domains were predicted by the TMHMM Server v2.0
([224]http://www.cbs.dtu.dk/services/TMHMM/). Protein conserved domains
were predicted by InterPro ([225]http://www.ebi.ac.uk/interpro/).
Best-matched homologs of planthopper species were aligned with the
ClustalX program v1.81^[226]54.
L. striatellus infestation and SA treatment
To investigate the effect of L. striatellus and SA on rice defense, the
4-5-leaf stage rice seedlings were sprayed with 0.5 μM SA (#84210,
Sigma-Aldrich, St. Louis, MO, USA) or infested by 5th instar L.
striatellus nymph (5 nymphs per plant, confined in a 5-cm plant stems
with a plastic cup). The treated plants were maintained in a climate
chamber at 25 °C, and samples were collected at indicated time points.
Quantitative real-time PCR analysis
Different tissue samples from carcasses (20), fat bodies (50), guts
(50), and salivary glands (80) were dissected from the 5th instar
nymphs in a phosphate-buffered saline (PBS) solution (137 mM NaCl,
2.68 mM KCl, 8.1 mM Na[2]HPO[4] and 1.47 mM KH[2]PO[4] at pH 7.4) using
a pair of forceps (Ideal-Tek, Switzerland). Similarly, testes (50) and
ovaries (20) were collected from adult male and female L. striatellus,
respectively. The number of insects in each sample was given in the
parentheses above. To extract RNA from N. benthamiana and rice, plants
were firstly grinded with liquid nitrogen. Then, samples were
homogenized in the TRIzol Total RNA Isolation Kit (#9109, Takara,
Dalian, China), and total RNA was extracted following the
manufacturer’s protocols. Afterwards, the first strand cDNA was
reverse-transcribed from RNA using HiScript II Q RT SuperMix (#R212-01,
Vazyme, Nanjing, China). qRT-PCR was subsequently run on a Roche Light
Cycler® 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany)
using the SYBR Green Supermix Kit (#11202ES08, Yeasen, Shanghai,
China). The PCR procedure was as follows, denaturation for 5 min at
95 °C, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The
primers used in qRT-PCR were designed using Primer Premier v6.0
(Supplementary Table [227]3). L. striatellus actin, O. sativa actin,
and N. benthamiana actin were used as internal controls, respectively.
The relative quantitative method (2^−ΔΔCt) was employed to evaluate the
quantitative variation. qRT-PCR results with a Ct value ≥35 were
regarded that the gene was not expressed in the sample. Three
independent biological replicates with each repeated twice were
performed.
RNA interference
The DNA sequences of target genes were amplified using the primers
listed in Supplementary Table [228]3, and cloned into pClone007 Vector
(#TSV-007, Tsingke, Beijing, China). The PCR-generated DNA templates
containing T7 sequence was used to synthesize the double-stranded RNAs
with a T7 High Yield RNA Transcription Kit (#TR101-01, Vazyme). RNA
interference experiment was conducted as previously described^[229]55.
Briefly, insects were anaesthetized with carbon dioxide for 5–10 s.
Then, dsRNA was injected into the insect mesothorax using FemtoJet
(Eppendorf-Netheler-Hinz, Hamburg, Germany). Afterwards, insects were
kept on the 4–5-leaf stage rice seedlings for 24 h and the living
insects were selected for further investigation. Silencing efficiency
was determined at 4th day post-injection using qRT-PCR method as
described above.
Insect bioassays
To perform survivorship analysis, a group of 30–40 treated insects (3rd
instar nymph) were treated with dsRNA and kept on 4–5 leaf stage rice
seedlings in a climate chamber. The mortality rates for each treatment
were recorded for ten consecutive days. Three independent replications
were performed. For honeydew analysis, a parafilm (Bemis NA, Neenah,
WI, USA) sachet was attached to the host plant stems, and the insects
(5th instar nymph) were confined in a sachet. At 24 h after feeding,
the accumulation of honeydew was measured by weighing the parafilm
sachet before and after feeding with an electronic balance (accuracy,
0.001 g; Sartorius, Beijing, China). At least 10 replicates were
performed for each treatment. For fecundity analysis, the newly emerged
adults were treated with dsRNA. One day later, the insects were paired
and allowed for oviposition for 10 days. Afterwards, the number of
hatched offspring was counted. At least 10 replicates were conducted
for each treatment.
Host choice test
The 4–5-leaf stage rice seedling was first placed in a glass tube, and
5 dsRNA-treated L. striatellus (5th instar) were allowed to feed on one
rice plant for 24 h. Thereafter, the insects were removed, and rice
plants pre-infested by different dsRNA-treated L. striatellus were
confined in a plastic cup (diameter, 6 cm; height, 10 cm), where a
release chamber was contained. Later, a group of 17 L. striatellus (4th
instar; wild-type, WT) were placed in the release chamber. The numbers
of insects settling on each plant were counted at 1, 3, 6, 12, 24, 36,
and 48 h, respectively. At least ten replicates were performed.
EPG recording analysis
The GiGA-8d EPG amplifier (Wageningen Agricultural University,
Wageningen, The Netherlands) with a 10 TΩ input resistance and an input
bias current less than 1 pA was used for EPG recording. Briefly, the
dsRNA-treated L. striatellus (5th instar) were reared on filter paper
with only water provided for 12 h. After anesthetizing by CO[2] for
10 s, a gold wire (Wageningen Agricultural University, diameter, 20 mm;
length, 5 cm) was applied in connecting insect abdomen and the EPG
amplifier with a water-soluble silver conductive glue (Wageningen
Agricultural University). The plant electrode was designed by inserting
a copper wire (diameter, 2 mm; length, 10 cm) into soils that were
planted with one rice plant. Later, EPG recording was conducted for 8 h
in a Faraday cage (120 cm × 75 cm × 67 cm, Dianjiang, Shanghai, China),
with the gain of the amplifier being set at 50× and the output voltage
being adjusted between −5V and +5 V.
The output data were analyzed by PROBE 3.4 (Wageningen Agricultural
University), and the insect feeding behaviors were classified into
nonpenetration (np), pathway duration (N1 + N2 + N3), phloem sap
ingestion (N4), and xylem sap ingestion (N5)^[230]48. At least 10
replicates were performed for each treatment.
Immunohistochemistry staining
To prepare insect tissues, salivary glands were dissected from L.
striatellus and fixed in 4% paraformaldehyde (#E672002, Sango
Biotechnology, Shanghai, China) for 30 min. To prepare salivary sheath
sample, the parafilm attached with salivary sheath was washed in PBS
and fixed in 4% paraformaldehyde for 30 min. To prepare plant tissues,
the rice plants infested by L. striatellus were collected and cut into
segments ~3 cm in length using a scalpel. Then, the short rice sheaths
were fixed in 4% paraformaldehyde and vacuumized at 4 °C. Afterwards,
the sheaths were blocked with Jung Tissue Freezing Medium (#020108926,
Leica Microsystems, Wetzlar, Germany) at −40 °C. Later, the blocks were
cut into 20 μm cross-sections using Cryostar NX50 (Thermo Scientific,
Waltham, MA), and fixed in 4% paraformaldehyde for the additional
30 min. The anti-LsSP1 serum, prepared by immunizing rabbits with
purified GST-LsSP1 proteins, was produced via the custom service of
Huaan Biotechnology Company (Hangzhou, China). The anti-OsOryzain
serum, prepared by immunizing rabbits with peptides VRMERNIKASSGKC and
DVNRKNAKVVTIDSY, was produced via the custom service of Genscript
Biotechnology Company (Nanjing, China). The anti-LsSP1 serum was
conjugated with Alexa Fluor™ 488 NHS Ester (#A20000, ThermoFisher
Scientific), while the anti-OsOryzain serum was conjugated with Alexa
Fluor™ 555 NHS Ester (#A37571, ThermoFisher Scientific) following the
manufacturer’s protocols. Thereafter, the insect/plant/parafilm samples
were incubated with the above fluorophore-conjugated serums overnight
at 4 °C with a dilution of 1:200, the actin dye phalloidinrhodamine
(#A22287, ThermoFisher Scientific) at room temperature with a dilution
of 1:500 for 30 min, and 4′,6-diamidino-2-phenylindole (DAPI) solution
(#ab104139, Abcam, Cambridge, USA). Finally, fluorescence images were
obtained using a Leica confocal laser-scanning microscope SP8 (Leica
Microsystems).
Preparation of protein samples
Salivary sheath samples and watery saliva samples were collected from
900 to 1000 nymphs as previously described^[231]14,[232]56. Briefly,
the 5th instar L. striatellus nymphs were transferred from the rice
seedlings into a plastic Petri plate. Approximately 300 μl diets with
2.5% sucrose were added between two layers of stretched Parafilm, and
the insects were allowed to feed for 24 h. Ten devices were used for
saliva collection, with each device containing 90–100 L. striatellus.
For the preparation of watery saliva samples, the liquid was collected
from the space between two layers of Parafilm. To prepare salivary
sheath samples, the upper surface of Parafilm with salivary sheath
firmly attached was carefully detached, and washed in PBS thrice. As
salivary sheath was difficult to dissolve, a lysis buffer of 4%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (#20102ES03,
Yeasen), 2% SDS (#A600485, Sango Biotechnology) and 2% DTT (#A100281,
Sango Biotechnology) was adopted for obtaining the solubilized salivary
sheath proteins under gentle shaking on an orbital shaker at room
temperature for 1 h according to previous description^[233]14,[234]56.
With this method, the majority of salivary sheath, although not all,
can be dissolved^[235]56. Since it was difficult to quantify the
protein concentration in saliva solution, the salivary sheath samples
and watery saliva samples was pooled to 50 μl using 3-kDa
molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device
(Millipore, MA, USA), respectively.
The rice apoplast was collected with Buffer A (consisting of 0.1 mol/L
Tris-HCl, 0.2 mol/L KCl, 1 mmol/L PMSF, pH 7.6) as previously
described^[236]57. Briefly, 5.0 g rice plants were vacuum infiltrated
with Buffer A for 15 min. Then, the remaining liquid on the surface was
dried with the absorbent paper, placed inside the 1-ml tips and
centrifuged in the 50-ml conical tubes at 1000 × g for 20 min. The
apoplastic solution was concentrated using 3-kDa molecular-weight
cutoff Amicon Ultra-4 Centrifugal Filter Device.
For the preparation of insect and plant samples, the insects/plants
were collected at indicated time points and homogenized in the RIPA
Lysis Buffer (#89900, ThermoFisher Scientific). To detect the secretion
of LsSP1 into rice plants, approximately one hundred 5th instar nymphs
were confined in the 2-cm stem and allowed to feed for 24 h. The outer
rice sheath was collected for western-blotting assay.
Western-blotting assay
The protein concentrations were quantified using a BCA Protein Assay
Kit (#CW0014S, CwBiotech, Taizhou, China) in line with the
manufacturer’s instructions. After the addition of 6× SDS loading
buffer, the protein samples were boiled for 10 min. Proteins were
separated by 12.5% SDS-PAGE gels and transferred to PVDF membranes.
Then, the blots were probed with anti-LsSP1 serum or anti-OsOryzain
serum diluted at 1:5000, followed by additional incubation with
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody
(1:10,000, #31460, ThermoFisher Scientific). Images were acquired by an
AI 680 image analyzer (Amersham Pharmacia Biotech, Buckinghamshire,
UK). The band intensities in immunoblot analyses were quantified using
ImageJ software v1.53e ([237]https://imagej.nih.gov/). To monitor the
equal protein loading, samples were further stained with Coomassie
brilliant blue (CBB). The full scan results of blots and gels were
provided in Supplementary Fig. [238]23 and Source Data file.
Identification and phylogenetic analysis of PLCP
The O. sativa PLCPs were investigated based on the procedure described
previously^[239]25. Briefly, amino acid sequences of 31 Arabidopsis
thaliana PLCPs^[240]58 were retrieved and used as queries to search for
PLCP homologs in the Rice Genome Annotation Project Database
([241]http://rice.plantbiology.msu.edu), with a cutoff e-value of
10^−5. The putative PLCPs were further validated by aligning to the
NCBI nr database. Thereafter, the structure and conserved domains of
PLCPs were analyzed by InterPro. Seven proteins predicted in Rice
Genome Annotation Project Database were incomplete, including
Os04g55650 ([242]NP_001389372), Os09g39160 ([243]BAD46641), Os09g39090
([244]XP_015611357), Os09g39170 ([245]BAD46642), Os09g39120
([246]XP_015611254), Os01g24570 ([247]BAD53944), and Os07g01800
([248]BAC06931). The complete sequences were retrieved from the NCBI
database by BLAST search, and the corresponding GenBank accessions were
provided in the brackets. For phylogenetic analysis, all PLCPs were
aligned with MAFFT v7.450, and the gaps were further trimmed using
Gblock v0.91b^[249]59. The substitution model was evaluated using
ModelTest-NG based on the default parameters^[250]60. Afterwards,
maximum likelihood (ML) trees were constructed using RAxMLNG v0.9.0
with 1000 bootstrap replications^[251]61.
Scanning electron microscopy
Insects were allowed to feed on rice plants or artificial diets for
24 h. The rice plant and parafilm attached with salivary sheath were
cut and washed with PBS. Later, SEM samples were attached to a stub and
dried in a desiccator under vacuum. After gold-sputtering, the samples
were observed by SEM TM4000 II plus (Hitachi, Tokyo, Japan). The length
of salivary sheath on the Parafilm membrane was measured from the top
to base of salivary sheath (Supplementary Fig. [252]4a), while the
number of salivary sheaths left on the rice surface was measured by
counting the ring-shaped salivary sheath structure (Supplementary
Fig. [253]4c) on 4-cm rice stem.
Agrobacterium-mediated plant transformation and diaminobenzidine staining
Details in Agrobacterium-mediated plant transformation in N.
benthamiana and diaminobenzidine staining of N. benthamiana leaves were
described in Supplementary Methods.
Protein–protein interaction assays
Details in the Y2H screening assay, Y2H point-to-point verification
assay, GST pull-down assay, BiFC assay, luciferase complementation
(LUC) assay, and OsOryzain-salivary sheath binding assay were described
in Supplementary Methods.
Generation of transgenic rice plants
To generate the oeSP1 plants, the coding sequence (without signal
peptide) was amplified and cloned into the binary expression vector
driven by a CaMV 35S promoter. The recombinant vector was introduced
into A. tumefaciens strain EHA105 by the heat transfer method.
Transgenic rice plants were generated through Agrobacterium-mediated
transformation. Briefly, rice seeds (cv. Nipponbare) were sterilized
with 75% ethanol for 1 min and 50% sodium hypochlorite for 20 min.
After washing in sterile water for three times, the sterilized seeds
were transferred onto NBi medium (N6 macro elements, B5 microelements,
B5 vitamin, 27.8 mg/L FeSO[4] · 7H[2]O, 37.3 mg/L Na[2]-EDTA, 500 mg/L
proline and glutamic acid, 300 mg/L casein hydrolyte, 2 mg/L
2,4-dichlorophenoxyacetic acid, 100 mg/L inositol, and 30 g/L sucrose)
for 20 days at 26 °C for callus induction. The induced calli were
incubated with Agrobacterium (OD[600] = 0.2) for 10 min, and then
cultured in NBco medium (NBi medium supplied with 100 µmol/L
acetosyringone, pH 5.5) for 3 days at 20 °C. After washing with sterile
water, the calli were transferred onto NBs medium (NBi medium supplied
with 500 mg/L cephamycin and 30 mg/L hygromycin) for 25 days.
Subsequently, the resistant calli were transferred onto NBr medium (NBi
medium supplied with 0.5 mg/L α-naphthalene acetic acid, 3 mg/L
6-benzylaminopurine, 500 mg/L cephamycin, and 30 mg/L hygromycin) for
shoot regeneration. The regenerated shoots were transferred into 1/2×
Murashige–Skoog medium for rooting. The transgenic plants were grown in
the greenhouse, and was confirmed by RT-PCR with reverse-transcribed
cDNA as the template using LsSP1-specific primers (Supplementary
Table [254]3). Two independent T3 homozygous overexpression lines
(Supplementary Fig. [255]17a) were used for subsequent experiments.
Evaluation of L. striatellus resistance in transgenic rice plants
The L. striatellus resistance in rice plants was scored as previously
described^[256]62,[257]63. Briefly, five rice seedlings were grown in a
10-cm-diameter plastic cup with a hole at the bottom. At the 4–5 leaf
stage, the seedlings were infested with L. striatellus nymphs (4th
instar; wild-type, WT) at a dose of 10 insects per seedling. After 20
days, the injury level of rice plants was checked, and the
identification standard was adopted for calculating the average injury
level^[258]62 (Supplementary Table [259]4). Four replicates were
performed for each line.
Performance of dsRNA-treated L. striatellus on transgenic rice plants
To investigate the performance of dsRNA-treated L. striatellus on
transgenic rice plants, 3rd instar nymph (for survivorship analysis),
4th instar nymph (for honeydew and EPG analyses), and newly emerged
adults (for fecundity analysis) were treated with dsGFP and dsLsSP1,
respectively. Insect bioassays for survivorship, honeydew, fecundity,
and EPG analyses were performed as described above. Two independent
homozygous overexpression/knockout transgenic lines were used.
Transcriptomic sequencing
The untreated rice plants or rice plants infested by dsLsSP1-treated L.
striatellus for 24 h were collected and homogenized in the TRIzol
Reagent (#10296018, Invitrogen, Carlsbad, CA, USA). Thereafter, total
RNA was extracted according to the manufacturer’s instructions, and the
RNA samples were sent to Novogene Institute (Novogene, Beijing, China)
for transcriptomic sequencing as previously described^[260]64. Briefly,
poly(A) + RNA was purified from 20 μg pooled total RNA by using
oligo(dT) magnetic beads. Fragmentation was implemented in the presence
of divalent cations at 94 °C for 5 min. Then, N6 random primers were
used for reverse transcription into the double-stranded complementary
DNA (cDNA). After end-repair and adapter ligation, the products were
amplified by PCR and purified using a QIAquick PCR purification kit
(Qiagen, Hilden, Germany) to create a cDNA library. The library was
sequenced on an Illumina NovaSeq 6000 platform. Thereafter, all
sequencing data generated were submitted to the NCBI Sequence Read
Archive under accession number PRJNA833487 and PRJNA815455.
Analysis of transcriptomic data
The output raw reads were filtered using the internal software, and the
clean reads from each cDNA library were aligned to the reference
sequences in Rice Genome Annotation Project Database using HISAT
v2.1.0^[261]65. The low-quality alignments were filtered by SAMtools
v1.7^[262]66. Transcripts per million (TPM) expression values were
calculated using Cufflink v2.2.1^[263]67. The DESeq2 v2.2.1^[264]68 was
adopted for analyzing the DEGs, and genes with log2-ratio > 1 and
adjusted p value < 0.05 were identified. To reveal overall differences
in gene expression patterns among different transcriptomes, R function
plotPCA (github.com/franco-ye/TestRepository/blob/main/PCA_by_deseq2.R)
and DNAstar v8.0^[265]69 were used to perform PCA analysis and
correlation analysis, respectively. KEGG enrichment analyses were
performed using TBtools software v1.0697^[266]70. In this software,
enriched P-values were calculated according to one-sided hypergeometric
test:
[MATH:
P=1−∑i=0m−<
mn>1MiN−M
n−i
Nn :MATH]
, with N represents the number of gene with KEGG annotation, n
represents the number of DEGs in N, M represents the number of genes in
each KEGG term, m represents the number of DEGs in each KEGG term.
Statistical analysis
The log-rank test (SPSS Statistics 19, Chicago, IL, USA) was applied to
determine the statistical significance of survival distributions.
Two-tailed unpaired Student’s t test (comparisons between two groups)
or one-way ANOVA test followed by Tukey’s multiple comparisons test
(comparisons among three groups) was used to analyze the results of
qRT-PCR, EPG, proteolytic activity, honeydew measurement, offspring
measurement, and host choice analysis. The exact p value of each
statistical test was provided in Source data file. Data were graphed in
GraphPad Prism 9.
Reporting summary
Further information on research design is available in the [267]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[268]Supplementary Information^ (3.9MB, pdf)
[269]Supplementary Data 1^ (36.5KB, xlsx)
[270]Supplementary Data 2^ (54.7KB, xlsx)
[271]Supplementary Data 3^ (386.8KB, xlsx)
[272]Supplementary Data 4^ (234.1KB, xlsx)
[273]Supplementary Data 5^ (182.7KB, xlsx)
[274]Reporting Summary^ (301.9KB, pdf)
[275]Peer Review File^ (3.1MB, pdf)
[276]41467_2023_36403_MOESM9_ESM.pdf^ (2.7KB, pdf)
Description of Additional Supplementary Files
Acknowledgements