Comparative transcriptome and histological analyses of wheat in response to phytotoxic aphidSchizaphis graminumand non-phytotoxic aphidSitobion avenaefeeding

Background

Infestation of the phytotoxic aphidSchizaphis graminumcan rapidly induce leaf chlorosis in susceptible plants, but this effect is not observed with the nonphytotoxic aphidSitobion avenae．However, few studies have attempted to identify the different defence responses induced in wheat byS. graminumandS. avenaefeeding and the mechanisms underlying the activation of chlorosis byS. graminum喂食。

Results

S. graminumfeeding significantly reduced the chlorophyll content of wheat leaves, and these effects were not observed withS. avenae．表达的转录组分析显示sion levels of genes involved in the salicylic acid, jasmonic acid and ethylene signalling defence pathways were significantly upregulated by bothS. avenaeandS. graminumfeeding; however, more plant defence genes were activated byS. graminum喂养比S. avenae喂食。The transcript levels of genes encoding cell wall-modifying proteins were significantly increased afterS. graminumfeeding, but only a few of these genes were induced byS. avenae．Furthermore, variousreactive oxygen species-scavenging genes, such as 66peroxidase(POD) and 8ascorbate peroxidase(APx) genes, were significantly upregulated afterS. graminumfeeding, whereas only 15PODand oneAPxgenes were induced byS. avenae喂食。The activity of four antioxidant enzymes was also significantly upregulated byS. graminum喂食。Cytological examination showed thatS. graminumfeeding induced substantial hydrogen peroxide (H₂O₂) accumulation in wheat leaves. The chlorosis symptoms and the loss of chlorophyll observed in wheat leaves afterS. graminumfeeding were reduced and inhibited by the scavenging of H₂O₂by dimethylthiourea, which indicated that H₂O₂plays important role in the induction of chlorosis byS. graminum喂食。

Conclusions

S. graminumandS. avenaefeeding induces the JA, SA and ET signalling pathways, butS. graminumactivated stronger plant defence responses thanS. avenae．S. graminumfeeding triggers strongROS-scavenging activityand massive H₂O₂production in wheat leaves, and the accumulation of H₂O₂induced byS. graminumfeeding is involved in the activation of chlorosis in wheat leaves. These results enhance our understanding of mechanisms underlying aphid-wheat interactions and provide clues for the development of aphid-resistant wheat varieties.

通过与昆虫相互作用超过一百illion years, plants have evolved complex and accurate defence mechanisms against herbivores. In response to herbivory, plants can perceive damage-associated molecular patterns (DAMPs) or herbivory-associated molecular patterns (HAMPs) in insect oral secretions and subsequently induce direct and indirect plant defence responses [1,2]。Direct defences involve the production and accumulation of plant defensive chemicals, such as plant secondary metabolites (PSMs), proteinase inhibitors, polyphenol oxidases and other defensive proteins, which are induced by herbivory and reduce herbivore performance [3,4]。Indirect defences include the synthesis and release of complex blends of volatiles that attract parasitoids and predators of the herbivores [5,6]。

Insects from different feeding guilds tend to elicit distinct plant defensive strategies of plants [7,8]。For example, leaf-eating beetles (Coleoptera) or caterpillars (Lepidoptera) cause extensive tissue damage during herbivory, which usually activates the jasmonic acid (JA)-mediated defence pathway in plants [9,10]。不同于叶咀嚼昆虫、半翅类昆虫s, such as aphids and whiteflies, have highly modified piecing-sucking mouthparts (stylets) that can penetrate the extracellular pathway and feed on the nutrients from phloem sap provided by sieve elements (SEs). Although the stylets puncture through most parts of plant cells during the probing track, these insects cause less damage to cells than leaf-chewing herbivores [11]。Many studies have demonstrated that hemipteran feeding results in the induction of the salicylic acid (SA)-dependent defence pathway in plants [12,13]。

The grain aphidSitobion avenaeand the greenbugSchizaphis graminum(Hemiptera: Aphididae) are considered two important pests of wheat and other cereals worldwide, as they suck phloem sap and serve as vectors of the barley yellow dwarf virus (BYDV), resulting in significant yield losses [14,15]。Aphids are classified as nonphytotoxic or phytotoxic according to the symptoms and damage caused by their feeding [16]。Similar to most aphid species,S. avenaeare nonphytotoxic, and no typical symptoms of plant damage are rapidly induced by their feeding processes. However, infestation byS. graminum, a phytotoxic species, can immediately induce obvious leaf chlorosis in susceptible plants, resulting in the deterioration of plant quality and even in plant death [17,18]。Several studies have characterized the plant defence responses induced by infestation ofS. avenaeandS. graminum．For example, Zhao et al. found thatS. avenaefeeding induces the expression of several genes involved in both the SA- and JA-mediated defence pathways in wheat [19], and Zhu-Salzman et al. demonstrated thatS. graminumfeeding strongly induces the expression of genes involved in the SA-dependent pathway in sorghum (Sorghum bicolor) but weakly increases the expression levels of JA-related defence genes, such as lipoxidase (LOX)and proteinase inhibitors (PIs) [20]。使用互补脱氧核糖核酸微阵列,几个成绩单with significantly different expression patterns were identified in sorghum seedlings afterS. graminumfeeding, and these herbivore-responsive genes were mainly associated with photosynthesis, the biosynthesis of defence molecules, cell wall fortification, oxidative bursts and stress [21]。

Previous studies have also shown thatS. graminumfeeding significantly increases the concentrations of amino acids, particularly essential amino acids, in the phloem of wheat and barley, and the enhancement of the nutritional quality of plants induced byS. graminumfeeding improves the aphid performance [22]。It has been proposed that leaf-senescence-like changes triggered byS. graminumfeeding are associated with the nutritional enhancement of host plants [18,23]。喂养造成的损害植物性毒素的蚜虫是usually observed in susceptible hosts; therefore, it has been hypothesized that the nutritional enhancement of plants derived from senescence-like feeding damage is a strategy used by phytotoxic aphids to potentially counteract the negative effects of induced plant resistance, which would eventually improve aphid fitness on the host [18]。

However, few studies have attempted to identify the molecular basis of the necrosis symptoms in wheat triggered by the phytotoxic aphidS. graminumand the different defence responses induced byS. graminumandS. avenae喂食。In this study, we integrated gene expression profiling through high-throughput RNA sequencing (RNA-Seq) with cytological examination to reveal the responses of wheat leaves toS. graminumandS. avenaefeeding, to compare the differences in the metabolic pathways affected by the two cereal aphids and to uncover the mechanism underlying the induction of damage symptoms byS. graminum．

Damage symptoms and changes in the chlorophyll content of wheat leaves afterS. avenaeandS. graminumfeeding

As shown in Fig.1a-c, no obvious damage symptoms were detected in leaves 48 h afterS. avenaeinfestation compared with leaves without aphid infestation, whereasS. graminumfeeding caused severe chlorosis in wheat leaves. Delayed fluorescence, which was used as a direct indicator of the chlorophyll content, was also measured in the wheat leaves 48 h after aphid infestation. As demonstrated in Fig.1d-f, the untreated leaves and theS. avenae-infested leaves exhibited strong signals of delayed fluorescence, whereas low signals of delayed fluorescence were detected in the wheat leaves infested withS. graminum, which suggested the occurrence of chlorophyll degradation.

The chlorophyll content of the leaves after aphid feeding was further investigated. As shown in Fig.2, no significant differences in the chlorophyll content were found between theS. avenae-infested and control plants. However, the chlorophyll content was significantly decreased to 0.46 ± 0.068 (F_2,6 = 10.494,P = 0.011) afterS. graminum喂食。

Transcriptome data from aphid-infested wheat leaves

The transcriptomes of wheat leaves infested with the two cereal aphid species were compared in this study. A total of 62.98 Gb of clean data were obtained from the nine leaf samples, and each of these samples contained ≥6.91 Gb with Q30 quality scores of ≥94.82% (Additional file1: Table S1). Subsequently, 83.5 to 94.3% of the clean reads from each sample were aligned onto the wheat reference genome and matched to either unique or multiple genomic locations (Table1).

Identification and functional annotation of DEGs

The gene expression levels of each replicate were assessed through principal component analysis (PCA). Replicates from the same group were clustered closely together, which suggested that the repeatability of each treatment group was satisfactory, and the samples from theS. avenae- andS. graminum-infested groups (Sa48h and Sg48h, respectively) clustered far from the control samples, which indicated that aphid feeding induced significant changes in gene expression (Fig.3). A total of 12,8195 transcripts were detected across all the samples (Additional file2: Data S1). Gene expression levels with an adjustedPvalue < 0.00001 and |Log₂Fold Change| ≥ 1 were selected as DEGs for further analysis. Forty-eight hours ofS. avenaefeeding significantly upregulated 1718 genes and significantly downregulated 172 genes in wheat leaves (Fig.4a). In addition, 7893 and 5098 genes were significantly upregulated and downregulated, respectively, after 48 h ofS. graminumfeeding (Fig.4b).

To investigate the differences in plant responses to infestation byS. graminumandS. avenae, the DEGs in wheat leaves induced by these aphids were also compared in our study. The results showed that the expression levels of 857 genes in wheat leaves were significantly upregulated by bothS. graminumandS. avenae喂食。Additionally, 11,046 and 1914 transcripts were specifically and significantly upregulated afterS. graminumandS. avenaefeeding for 48 h, respectively (Fig.5a). In contrast, a total of 128 transcripts were significantly downregulated after bothS. graminumandS. avenaeinfestation, and 7036 and 861 genes were only significantly downregulated afterS. graminumandS. avenaefeeding, respectively (Fig.5b). This finding suggested that the global response of wheat toS. graminumfeeding is distinct from that of wheat toS. avenae喂食。

All the DEGs were subjected to GO term and KEGG pathway analyses to identify the major DEG-associated metabolic pathways. The top 30 enriched GO terms and 20 most enriched KEGG pathways are shown in Fig.6and Figure S1 (Additional file3). As shown in Fig.6, within the biological process category, the DEGs induced byS. graminumwere mainly enriched in the electron transport, small molecule metabolic process and carbohydrate metabolic process terms, and the DEGs induced byS. avenaewere mainly enriched in protein phosphorylation, protein modification process and phosphorus metabolic process. Within the molecular function category, the largest proportion of DEGs induced byS. graminumwas enriched in the catalytic activity and oxidoreductase activity terms, and the majority of the DEGs activated byS. avenaewere enriched in the catalytic activity, protein kinase activity and phosphotransferase activity.

Transcripts related to photosynthesis, sucrose and starch metabolism and nitrogen metabolism

S. graminumfeeding negatively affected the photosynthesis process of wheat, and many genes associated with light-harvesting and photosystem-associated genes, such as chlorophyll a-b binding proteins, ferrochelatase, and photosystem I and II proteins, were significantly downregulated (Table2). The expression levels ofribulose bisphosphate carboxylase oxygenase(RuBisCO) andcarbonic anhydrasegenes with roles in the Calvin cycle were also significantly reduced afterS. graminum喂食。However, few genes involved in photosynthesis were significantly regulated in theS. avenae-infested plants. The transcriptional profiles of some genes involved in sucrose and starch metabolism were also investigated. Thesucrose synthase 3gene, onetrehalose-6-phosphate synthasegene and sixbeta-glucosidasegenes were significantly downregulated inS. graminum-andS. avenae-infested leaves, and the transcript levels ofsucrose-phosphatasegenes were significantly upregulated in wheat leaves infested withS. graminumandS. avenae．S. graminumfeeding but notS. avenaefeeding also significantly affected nitrogen metabolism. The transcript levels ofnitrate reductasein the leaves were strongly downregulated byS. graminum, andglutamate dehydrogenasewas significantly upregulated in wheat leaves infested withS. graminum．However, few genes involved in nitrogen metabolism were modulated byS. avenae喂食。

Effects ofS. graminumandS. avenaefeeding on transcripts related to the SA, JA, and ET signalling pathways involved in plant defence

To characterize how plant defence responses are modulated in response toS. graminumandS. avenaefeeding, genes known to be involved in the SA, JA, and ET-defence pathways were examined [24]。The transcriptome data in Table3showed that 21PALgenes involved in SA biosynthesis were significantly upregulated in response toS. graminumandS. avenae喂食。Furthermore, 13PRgenes responding to SA were significantly upregulated byS. graminumandS. avenae喂食。Additionally, underS. graminumandS. avenaefeeding, oneAOC(3.7-fold), threeAOS(2.7- to 7.9-fold) and fiveLOX(2.8- to 8.8-fold) genes involved in JA biosynthesis were significantly upregulated, and the expression levels of threePIgenes, which are JA-responsive defence genes, significantly increased. TwoACS(5.1 to 7.8-fold) and threeACO(4.0 to 5.0-fold) genes, which are involved in the ET signalling pathway, and 11 genes encoding ethylene-responsive transcription factors involved in ET biosynthesis were significantly upregulated in response toS. graminumfeeding, but only oneACO(4.4-fold) gene and two genes encoding ethylene-responsive transcription factors (1.8-fold and induced) were upregulated afterS. avenae喂食。

Although the transcript levels of some defence genes were significantly upregulated in response to bothS. graminumandS. avenaefeeding, a higher number of DEGs involved in SA-, JA-, and ET-mediated defence pathways were induced byS. graminum喂养比byS. avenaefeeding (Additional file4: Data S2). For example, 37PRgenes (downstream of SA) and 10PIgenes (downstream of JA) were significantly upregulated in response toS. graminumfeeding, but only 17PRgenes and fourPIgenes were significantly upregulated in response toS. avenae喂食。Additionally,S. graminumfeeding induced greater fold changes in these two types of genes thanS. avenaefeeding, which indicated that the former triggered a stronger defence response than the latter.

Effects ofS. graminumfeeding on transcripts associated with plant cell wall modification proteins (PCMDPs) in wheat leaves

S. graminumfeeding induced the expression of many genes encoding enzymatic or non-enzymatic proteins related to plant cell wall dynamics (Table4). For example, the transcript levels of callose synthases were significantly upregulated afterS. graminumandS. avenae喂食。The transcript levels of 19PGs(1.92 to 6.45-fold; induced) and fourPEMs(3.37 to 3.97-fold; induced) were significantly increased after 48 h ofS. graminumfeeding, but noPGsorPEMswere significantly induced in response toS. avenae喂食。Similarly, the transcript levels of six genes encoding beta-expansin, which is a non-enzymatic protein that plays important roles in cell wall loosening, were significantly upregulated (5.30 to 8.70-fold; induced) in wheat leaves infested withS. graminum, but noexpansingenes were significantly regulated after 48 h ofS. avenae喂食。

Effects ofS. graminumandS. avenaefeeding on the transcript levels and activities of antioxidant enzymes involved in ROS scavenging in wheat leaves

In plants, herbivore attacks usually trigger oxidative responses [25]。Plants possess a battery of ROS scavengers, such as POD, SOD, and CAT enzymes, and these enzymes can protect cells from oxidative damage [26]。As shown in Fig.7, 74PODswere significantly up- or down- regulated in response toS. graminumfeeding, and 66 of thesePODs明显upregulated. However, only 15PODs明显upregulated byS. avenae喂食。Similarly, the expression levels of 12APxgenes were significantly modulated byS. graminumfeeding, but the expression levels of only twoAPxgenes were significantly affected byS. avenae喂食。Additionally, fiveCAT, eightSODand sevenglutathione peroxidase(GPx) genes were significantly regulated byS. graminumfeeding, but not byS. avenaefeeding (Additional file5: Data S3). The increased number of ROS scavengers induced byS. graminumfeeding suggested thatS. graminumfeeding induces stronger oxidative stress in wheat leaves thanS. avenae．

Compared with the control, the activity of POD was significantly increased after 24 h (t₄ = − 4.387,P = 0.012) ofS. graminumfeeding and reached a peak at 48 h (t₄ = − 9.981,P = 0.001) (Fig.8). The activity of POD in leaves was also significantly increased 72 h afterS. avenaefeeding (t₄ = − 3.353,P = 0.028). Furthermore, the activities of SOD, CAT and APx were significantly increased after 24 h ofS. graminumfeeding (t₄ = − 12.295,P < 0.001; t₄ = − 2.789,P = 0.049; t₄ = − 7.761,P = 0.001), whereasS. avenaefeeding had no significant effects on the activities of SOD, CAT and APx (t₄ = − 1.560,P = 0.194; t₄ = − 0.600,P = 0.581; t₄ = 0.048,P = 0.964).

Cytological examination of callose deposition and ROS accumulation in aphid-infested leaves

To detect whether callose was deposited at the feeding sites, aphid-infested leaves were stained with aniline blue. As shown in Fig.9, no callose deposits were observed in vascular tissues without aphid infestation (Fig.9a). However, inS. avenae-andS. graminum-infested tissues, callose deposits were clearly detected as bright blue fluorescence directly at the feeding sites (Fig.9b and c).

H₂O₂accumulation has been shown to be induced by wounding and by pathogen and herbivore attacks in plants and is involved in plant defence responses as a signal molecule [27]。To record the accumulation of H₂O₂after aphid infestation,S. avenae- andS. graminum-infested leaves were examined after cytological staining with DAB, which was used to detect the production of H₂O₂．As shown in Fig.9d-e, no obvious DAB staining was observed in the non-infested leaves, and a small brown-stained area was detected in the wheat leaves afterS. avenaefeeing. However, H₂O₂was clearly detected in the areas ofS. graminumfeeding, which indicated thatS. graminumfeeding induced a massive accumulation of H₂O₂(Fig.9f). The H₂O₂contents in the feeding sites of wheat leaves afterS. avenaeandS. graminuminfestations were also examined. As indicated in Fig.10, the concentration of H₂O₂in wheat leaves infested withS. graminum(143.19±31.15μg摩尔^− 1FW; F_2,6 = 7.345;P = 0.024) was significantly higher than that inS. avenae-infested and control leaves. In contrast,S. avenaefeeding had no significant effects on the H₂O₂content compared with the control (Fig.10). The changes in the H₂O₂content in response to aphid feeding were consistent with the DAB staining results.

Scavenging of H₂O₂using DMTU reducesS. graminumfeeding-induced damage on wheat leaves

To further investigate the role of H₂O₂accumulation on the damage induced byS. graminumfeeding, wheat seedlings infested with aphids were treated with 5 mM DMTU (an H₂O₂scavenger). The DAB staining results shown in Fig.11demonstrated that DMTU treatment inhibited theS. graminumfeeding-induced production of H₂O₂in wheat leaves and the symptoms of damage in wheat leaves caused byS. graminum喂食。延迟荧光和叶绿素content were also assessed, and the results showed that the DMTU-treated infested leaves showed decreased chlorophyll degradation and that the chlorophyll content in the DMTU-treated leaves was significantly higher than that in the non-DMTU-treated leaves infested withS. graminum(F_{2, 6} = 13.93,P = 0.0056).

A previous study showed thatS. graminumfeeding led to obvious feeding damage and the loss of chlorophyll in aphid-susceptible winter wheat accession Beijing 837 [18]。Similarly, serious chlorosis symptoms were observed on another winter wheat accession, Zhongmai 175, after 48 h ofS. graminumfeeding in this study, and this effect was also accompanied by a significant reduction in the total chlorophyll content of the wheat leaves, further demonstrating the phytotoxic effects ofS. graminumon susceptible wheat plants. To further compare the similarities and differences between the responses toS. graminumandS. avenaefeeding at the molecular level, a comparative transcriptome analysis of wheat leaves after aphid feeding was performed. We found that more than 20,000 genes were significantly regulated in wheat infested withS. graminum, but only 1700 genes were significantly modulated after 48 h ofS. avenaefeeding, which indicated that the physiological changes induced byS. graminumare notably different from those induced byS. avenaeand that various metabolic pathways are involved in the development of damage caused byS. graminum喂食。Moreover, many genes involved in plant photosynthesis were strongly downregulated afterS. graminumfeeding, and this finding provides molecular evidence showing that chlorosis is induced byS. graminum．

S. graminumfeeding induces stronger plant defence responses thanS. avenae

Piercing-sucking hemipteran insects, such as aphids and whiteflies, mainly induce SA-mediated defence signal pathways [28,29]。However, some studies have also demonstrated that genes involved in both the JA and SA defence response pathways, such asLOX,PIs,PAL, andPR1, are significantly upregulated in response to aphid feeding [20,30,31]。Similarly, we found that bothS. avenaeandS. graminumfeeding significantly increased the expression levels of genes related to the SA, JA and ET signalling pathways. Plant defence responses activated by aphids are closely associated with the plant species, aphid density and infestation time [32,33]。In the future, we will further identify the wheat defence responses induced byS. graminumandS. avenaefeeding under various aphid densities and feeding periods. Although both of these cereal aphids induced both SA-, JA- and ET-dependent defence pathways,S. graminumfeeding induced the expression of more genes involved in plant defence pathways in this study. For example, 30PALgenes were upregulated in response toS. graminumfeeding, but only fourPALgenes were upregulated in response toS. avenae喂食。The transcript levels of five ET-responsive genes were upregulated in response toS. graminum,but only one was modulated in response toS. avenae喂食。Zhang et al. demonstrated that the fold changes in the expression levels ofPRgenes and the SA contents in wheat leaves were significantly greater afterS. graminum喂养比afterS. avenaefeeding [22]。Argandoña et al. also suggested thatS. graminuminduced more ethylene production than the non-phytotoxic aphidRhopalosiphum padi[34]。The stronger defence responses activated byS. graminum喂养比byS. avenaefeeding might be responsible for the induction of chlorosis in wheat.

Genes encoding plant cell-modifying proteins are significantly upregulated in response toS. graminumfeeding

Multiple modifications can be triggered in cell walls in response to microbial and insect attack [35]。Callose deposition in crop plants is observed in response to biotrophic fungal infection at papillae sites and in sieve elements in response to aphids [36,37]。It has been proposed that callose deposition impedes fungal attacks at the sites of attempted penetration in epidermal cells and thereby supports pathogen resistance [38]。The transcript levels of callose synthases were significantly upregulated in response toS. graminumandS. avenae喂食。Obvious callose accumulation was also observed in wheat leaves afterS. avenaeandS. graminum喂食。However, the role of callose in aphid-plant interactions remains unknown. It has been hypothesized that callose deposition induced by aphids is involved in the sealing of the sieve pores as a phloem defence mechanism that impedes mass flow and prevents the flow of nutrients to piercing-sucking insects [39]。

Many studies have demonstrated that the damage symptoms induced by pathogens and herbivores in plants are caused by the secretion of plant cell wall enzymes during the process of pathogen infection and miridLygus hesperusfeeding [40,41]。Additionally, the activities of PEMs and PGs have also been detected inS. graminumwatery saliva, and the injection of these commercial enzymes in plant leaves causes damage symptoms similar to those induced byS. graminumfeeding [42,43]。However, pectinase activity has also been detected in the saliva of non-phytotoxic aphids, such asS. avenaeandA. pisum[43,44]。Transmission electron microscopy has shown that stylets predominantly penetrate between the layers of cellulose fibres and not via the middle lamella pectin layer [45]。The role of the saliva pectinases ofS. graminumin the induction of chlorosis remains unclear. Interestingly, in our study, many plant-derived enzymes and proteins involved in plant cell wall modifications, such as PGs, PEMs and expansins, were induced byS. graminumfeeding, but none were induced byS. avenae喂食。The upregulation of PG and PEM activity might result in the degradation of the cell wall around aphid feeding sites. Plant cells exploit complicated mechanisms for sensing the loss of cell wall integrity (CWI) during biotic stress and activate a variety of defence responses [46]。For instance, the production of oligogalacturonic acid (OGA) fragments derived from the degradation of plant cell walls has been shown to trigger oxidative bursts, hypersensitive responses (HR), and other downstream defence responses in many plant species as a host-derived DAMP [47,48], and these effects might further promote the induction of damage symptoms. In addition, ethylene production has been shown to be involved in the induction of plant cell wall-modifying proteins and the death of plant tissues. The role of PCMDPs and the ethylene pathway in the induction of chlorosis symptoms byS. graminumneeds to be further investigated.

S. graminumfeeding induces strong ROS-scavenging activity in wheat leaves

Aphid feeding usually leads to oxidative stress in host plants [49]。Oxidative stress is controlled by cellular antioxidant mechanisms in which multiple enzymatic scavengers, such as POD, APx, and CAT, are utilized by the cell to limit damage from reactive oxygen species [50,51]。The transcriptomic and enzymatic results showed that bothS. graminumandS. avenaefeeding increased the transcript levels and enzyme activities of ROS scavengers in wheat leaves. However, the expression levels and activity of antioxidants, particularly POD, induced byS. graminumfeeding were notably higher than those induced byS. avenae, which suggested thatS. graminuminfestation results in strong oxidative stress and substantial H₂O₂accumulation. Although ROS scavengers were significantly upregulated in response toS. graminuminfestation, ROS production can exceed the cellular antioxidant capacity, resulting in oxidative damage to cellular components and cell death in leaves.

Induction of high H₂O₂accumulation byS. graminumfeeding is involved in leaf chlorosis

H₂O₂is involved in the activation of HR, which is characterized by the rapid death of cells in the region surrounding the site of pathogen infection site [52,53]。To further investigate the roles of H₂O₂in the induction of feeding damage caused byS. graminumfeeding, the accumulation of H₂O₂in wheat leaves was detected. We found thatS. graminumfeeding induced the obvious accumulation of H₂O₂at feeding sites, butS. avenaefeeding had no significant effects on H₂O₂production．In addition, seedlings treated with the H₂O₂scavenger DMTU showed reductions in the chlorosis symptoms and chlorophyll loss triggered byS. graminum喂食。These results demonstrate that H₂O₂accumulation plays important roles in the induction of chlorosis in wheat leaves in response toS. graminum喂食。

蚜虫唾液是参与引进ion of plant defence responses, and the eliciting activity of watery saliva of other aphid species such asM. persicaeandS. avenaehas been investigated [54,55]。The transient overexpression of Mp10, a salivary protein ofM. persicae,induces plant defence responses and obvious chlorosis inN. benthamiana[56]。Specific elicitors or pathogen-like toxins inS. graminumsaliva are likely involved in the induction of chlorosis. Comparative analyses of the salivary proteomes of four differentially virulentS. graminumbiotypes revealed six salivary proteins with significant proteomic variation, and these proteins might thus be involved in the induction of feeding damage in plants [57]。Further research is required to identify the virulence factors in the salivary proteins ofS. graminumand the mechanism underlying the induction of chlorosis.

In summary, the transcriptomic profiling of wheat performed in this study revealed similarities and differences among the responses of wheat to feeding by the phytotoxic aphidS. graminumand the non-phytotoxic aphidS. avenae．Both aphids induced the JA, SA and ET signalling pathways, butS. graminumtriggered stronger plant defence responsesand greater ROS-scavenging activitythanS. avenae．A cytological analysis showed that aphid feeding induced callose deposition in wheat leaves and that substantial H₂O₂accumulation was induced byS. graminum喂食。Our results also demonstrated that H₂O₂plays vital roles in the induction of chlorosis in wheat leaves in responses toS. graminum喂食。Our future studies will focus on the mechanisms of H₂O₂accumulation induced byS. graminumfeeding and the roles of salivary proteins ofS. graminumin the induction of chlorosis symptoms in wheat.

Plants and aphids

Seeds ofTriticum aestivumvar. Zhongmai 175 were germinated in distilled water for 3–4 days at a temperature of 25 ± 1 °C in a Petri dish. Healthy seedlings of similar sizes were planted in 7.2 × 7.2 cm plastic plots filled with organic soil and grown under controlled environmental conditions in climate chambers with a temperature of 20 ± 1 °C, a 40–60% relative humidity and a 14-h-light/10-h-dark photoperiod. Clones ofS. graminumandS. avenaewere maintained on the wheat plants (Zhongmai 175) as described previously [18]。

Aphid infestation

At the two-leaf stage (12-day old plants), 20 apterous adultS. graminumorS. avenaewere confined on the first leaf of wheat seedlings using a clip cage as described previously [18]。New-born nymphs produced by aphid adults were carefully removed every 12 h using a brush. After 48 h of feeding, all the aphids were removed, and leaf tissues of approximately 2.5 × 2.5 cm from the aphid feeding sites of each plant were harvested flash frozen with liquid nitrogen and stored at − 80 °C until further processing for RNA extraction. Detection of delayed fluorescence and histological staining were conducted immediately after sample collection. Three leaf sections covering the aphid feeding sites were collected from three independent plants and pooled to form one biological replicate. Three biological replicates were performed for each treatment.

Changes in chlorophyll levels in wheat leaves after aphid infestation

Delayed fluorescence is associated with extremely weak light emitted by chlorophyll molecules in plants and can reflect the chlorophyll content, providing a powerful tool for studying stress reactions in plants. The chlorophyll content in wheat leaves were each infested by 20 aphids as described above was first detected using the NightShade LB 985 In vivo Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany). After 48 h of aphid feeding, the leaves were cut and immediately illuminated for 30 s with an LED panel. After the light was switched off, the delayed fluorescence was measured immediately using the NightShade system. The exposure time was set to 30 s using 4-by-4 pixel binning. The total chlorophyll content in wheat leaves after aphid infestation was also examined using a Chlorophyll Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. In brief, 0.1 g of fresh leaf tissues was ground to a fine powder and extracted with 2 mL of 80% acetone (v/v) at 4 °C for overnight. The homogenate was centrifuged at 4000 g for 10 min at 4 °C, and the supernatant was used for the chlorophyll assay. The amounts of chlorophyll were detected spectrophotometrically, by reading the absorbance at 645 and 663 nm (DU800, Beckman, USA), and then calculated as described previously [58]。

RNA preparation and sequencing library construction

Wheat leaves were first infested with aphids for 48 h as described above. The total RNA from the wheat leaves was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer’s recommended protocol. The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., USA), and the RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA integrity number (RIN) ≥ 7.0 were used in the subsequent analysis. Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions and were sequenced on the Illumina sequencing platform (Illumina HiSeq 4000), which generated 150-bp paired-end reads were generated.

RNA sequencing and data analysis

The raw data (raw reads) were filtered to obtain high-quality reads by removing the reads containing adaptor sequences, more than 3% ambiguous bases (noted as N) or more than 50% low-quality bases (Phred quality score Q < 30). The resulting high-quality clean reads were then mapped to the reference genome (https://plants.ensembl.org/Triticum_aestivum/Info/Index) using TopHat2 with the default values [59]。The fragments per kilobase of exon model per million mapped reads (FPKM) data were used to estimate the transcript expression levels in all the samples, and the genes with more than 1 FPKM in at least one sample of wheat leaves were used for further analysis [60]。The differentially expressed genes (DEGs) between the control and treated samples were screened using DESeq based on the following criteria: adjustedpvalue (qvalue) threshold < 0.00001 and |log₂FC| ≥ 1. A PCA of nine different leaf samples was performed based on pairwise comparisons using the DESeq package of R [61]。GO enrichment and KEGG pathway enrichment analyses of the DEGs were performed using R based on the hypergeometric distribution [62]。To annotate the functions of the transcripts, the unigenes were blasted against the Nr database using the BLAST programme with anE-value ≤1e-5.

Assays of antioxidant enzymes in wheat leaves after aphid infestation

The leaves were infested with 20 apterous adultS. graminumorS. avenaeas described previously. The activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APx) in the wheat leaves at different time points after the aphid infestation (12 h, 24 h, 48 h and 72 h) were examined using corresponding kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, 0.2 g of fresh leaf tissues was ground with 1.5 mL of ice-cooled 50 mM Na-phosphate buffer (pH = 7.8) containing 0.1 mM EDTA and 1.0% (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 15,000 g for 30 min at 4 °C, and the supernatant was immediately collected for further enzyme assays. The activities of POD, SOD, CAT, and APx were measured by following the changes in absorbance at 470 nm, 560 nm, 240 nm and 290 nm, respectively, according to a previous study [63]。

Detection of H₂O₂and callose accumulation in wheat leaves after aphid infestation

The detection of H₂O₂在小麦叶片3 ' -diaminobenzidine (DAB)污渍ing was performed according to the histochemical methods described by Wang et al. [64] with some modifications. In brief, leaf segments previously infested with 20 apterous adults ofS. avenaeorS. graminumwere immersed in 1 mg mL^− 1DAB solution (10 mmol L^− 1Na₂HPO₄, pH 3.8), and incubated in the dark overnight at room temperature. Then, the leaves were decolorized in boiling 95% ethanol for 10 min and hyalinized in saturated chloral hydrate. The stained leaves were imaged using an Olympus BX-63 microscope (Olympus Corporation, Japan). The endogenous H₂O₂content in the wheat leaves after aphid feeding was determined using the protocols reported by Ferguson et al. [65]。For the visualization of callose, the leaves were first fixated, destained overnight in 1:3 acetic acid/ethanol (v/v) solution and washed in 150 mM K₂HPO4 for 30 min. The leaves were subsequently incubated for 6 h with 150 mM K₂HPO4 and 0.01% aniline blue for staining, and the callose depositions were observed and photographed with an Olympus SZX-16 fluorescence microscope (Olympus Corporation, Japan) using a DAPI filter.

Wheat seedlings treated with DMTU solution

The leaves of wheat seedlings were treated with 5 mM dimethylthiourea (DMTU, a scavenger of H₂O₂) solution or deionized water (control) for 24 h and then infested with 20 apterous adults ofS. graminumfor 48 h. Assessments of DAB staining and delayed fluorescence analyses and an assessment of the chlorophyll content of the wheat leaves were then performed as described previously.

Statistics analysis

All the data were analysed using SPSS Statistics 20.0 software (SPSS Inc., Chicago, IL., USA), and the differences between or among groups were examined through an independent-samplest-test or one-way analysis of variance (Duncan).Pvalues less than 0.05 were considered statistically significant.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

ACO:

1-aminocyclopropane-1-carboxylate oxidase 1

ACS:

1-aminocyclopropane-1-carboxylate synthase

AOC:

Allene oxide cyclase

AOS:

Allene oxide synthase

APx:

Ascorbate peroxidase

BYDV:

Barley yellow dwarf virus

CAT:

Catalase

CWI:

Cell wall integrity

DAB:

3′-diaminobenzidine

DAMPs:

Damage associated molecular patterns

DEGs:

Differentially expressed genes

DMTU:

Dimethylthiourea

ET:

Ethylene

EXP:

Beta-expansin

FDR:

Adjustedpvalue

FPKM:

Fragments per kilobase of exon model per million mapped reads

GPx:

Glutathione peroxidase

H₂O₂:

Hydrogen peroxide

HAMPs:

Herbivory associated molecular patterns

HR:

Hypersensitive response

JA:

Jasmonic acid

LOX:

Lipoxygenase

MeJA:

Methyl jasmonate

OGA:

Oligogalacturonic acid

PAE:

Pectin acetylesterase

PAL:

Phenylalanine ammonia-lyase

PEM:

Pectinesterase

PG:

Polygalacturonase

PIs:

Proteinase inhibitors

POD:

Peroxidase

PR protein:

Pathogenesis-related protein

RNA-Seq:

High-throughput RNA sequencing

ROS:

Reactive oxygen species

RuBisCO:

Ribulose bisphosphate carboxylase oxygenase

SA:

Salicylic acid

SEs:

Sieve elements

SOD:

Superoxide dismutase

我们要感谢技术员Yanxia女士刘for aphid rearing in our laboratory.

This study was funded by the National Natural Science Foundation of China (31871979, 31901881), National Key R&D Plan in China (2017YFD0201701, 2016YFD0300701), and the Cooperation Project between Belgium and China from MOST (2014DF32270). The funding organizations played no role in the design of study and collection, analysis, and interpretation of data and in writing the manuscript.

Affiliations

1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, People’s Republic of China

   Yong Zhang, Yu Fu, Jia Fan, Qian Li & Julian Chen

2. Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liège, B-5030, Gembloux, Belgium

   Frédéric Francis

Contributions

JLC and YZ designed the experiment, YZ, YF and JF performed the experiments, YZ, YF and QL analyzed the data, JLC and FF provided funding, YZ, JLC and FF wrote the first drafts of the manuscript. All authors critically read and approved the manuscript.

Corresponding author

Correspondence toJulian Chen．

Ethics approval and consent to participate

The seeds of wheat, Zhongmai 175 were bred by institute of Crop Sciences, Chinese Academy of Agricultural and Sciences and bought from Henan Shengyuan Seed Industry Technology Co., Ltd., Xuchang, China. Cereal aphidsSitobion avenaeandSchizaphis graminumused in our study were collected in Langfang, Hebei Province, China, and has been rear in our greenhouse for more than 9 years. The authors declare that all the experiments performed in this study comply with the institutional, national, or international guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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