Genes related to sucrose metabolism
Sucrose has a central position in plant metabolism as the first free sugar formed during photosynthesis [23, 24] and the major form of translocated sugar in the phloem. Soluble sugars, such as sucrose, glucose and fructose, can be donors of carbon skeletons for secondary metabolism and signaling molecules that regulated the expression of genes encoding flavonoid biosynthesis enzymes [19, 26]. Sucrose metabolism plays an important role in the defense against insect herbivores, such as aphids. In our study, as shown in Fig. 5A, the level of soluble sugar increased after 6 h of infestation and reached a maximum at 24 h in the Cm / As plants. At the same time point, a higher concentration of soluble sugar was observed in Cm / As than in Cm / Cm.
The multiple roles of plant sucrose transporters, especially the central role of sucrose loading into the phloem, suggest that sucrose transport is strongly regulated by biotic stresses [27-29]. In the present study, nine genes involved in the sucrose metabolism pathway were differentially expressed between Cm / As and Cm / Cm. As shown in Fig. 5B, three INV genes (Cluster-21353.120200, Cluster-21353.176307 and Cluster-21353.133225), one PYG gene (Cluster-21353.277981) and one PGM gene (Cluster-21353.147957) were strongly upregulated in Cm / As (B1) compared to Cm / Cm (A1) at 6 h, and these genes have been shown to be involved in glucose synthesis (Fig. 5C). In addition, there were four downregulated genes in B1 compared with A1, including two OTS genes (Cluster-21353.199573 and Cluster-21353.175120) and two BPGM genes (Cluster-21353.178805 and Cluster-21353.224391), which have been shown to be involved in glucolysis (Fig. 5C). The results suggested that grafting chrysanthemum onto A. scoparia W. accelerated the synthesis of soluble sugars, which increased the carbon metabolism burden of aphids and thus prevented aphids from feeding on the leaves. The details of these genes are shown in Additional file 2: Table S1. These genes may play an important role in promoting the synthesis of soluble sugars at the early stage in response to M. sanbourni infestation in the Cm / As seedlings.
Genes related to secondary metabolism
Secondary metabolites, for example, flavonoids, terpenes, phenolics and alkaloids, have antixenotic or antibiotic properties and thus function in the defense against insect herbivores, such as aphids . Among secondary metabolites, flavonoids are responsible for many key functions, which are critical for plant survival . Flavonoids are catalyzed by amount of enzymes, such as PAL, which has been studied in plant responses to biotic and abiotic stresses . In the present study (Fig. 6C), the activity of PAL in Cm / As was significantly increased by aphid infestation and higher than that in Cm / Cm, which promoted the accumulation of flavonoids. Studies on insect-plant interactions have revealed important contributions of flavonoids . In Vigna , there was a positive relationship between resistance or susceptibility properties against aphids and the flavonoid glycoside content. The content of flavonoids in susceptible lines was lower than that in resistant lines . In the present study, after A. sanbourni infestation, flavonoid accumulation was detected in the leaves of Cm / As and Cm / Cm. The level of flavonoids increased after 6 h of infestation, and a higher concentration of flavonoid was observed in Cm / As than in Cm / Cm (Fig. 6B). Additionally, genes related to flavonoid synthesis were also identified. Eight genes were differentially expressed between Cm / As and Cm / Cm (Fig. 6D-E ), including one CHS gene (Cluster-25249.0), which was downregulated in B1 compared to B0, and one malonyl-CoA gene (Cluster-21353.290401), which was strongly upregulated in B1 compared to A1 and may play an important role in Cm / As defense towards aphid feeding at the early stage of infestation. In addition, three flavonoid 3'-monooxygenase (F3'H) genes (Cluster-21353.144221, Cluster-21353.9823 and Cluster-21353. 344000), two flavonoid 3'5'-hydroxylase (F3'5'H) genes (Cluster-21353.241984 and Cluster-21353. 339941) and one flavonoid 3-O-glucosyltransferase gene (Cluster-21353.169006) were strongly upregulated in B2 compared to A2, which could play an important role in Cm / As defense towards aphid feeding in the later stage of infestation (Fig. 6D).
In addition, among secondary metabolites, alkaloids act as a defense against insects and other herbivores [35, 36]. For example, the reduced alkaloid content in sweet lupins led to a high susceptibility to insect herbivores, e.g., aphids [37-39]. In this study, only two alkaloid synthesis-related genes, STR genes (Cluster-21353.170595 and Cluster-21353.170592), were detected in Cm / As and Cm / Cm, both of which were downregulated at 96 h after aphid infestation.
Plants produce a vast array of volatiles that mediate their interaction with the environment and that consist of terpenoids and isoprenoids, which are synthesized through the condensation of C5 isoprene units. Monoterpenes and sesquiterpenes represent the C10 and C15 terpene classes, respectively. These compound classes have typical characteristics, such as volatility, flavor/aroma, and toxicity, and hence play important roles in plant defense and pollinator attraction . A previous study found that the increased content of monoterpenoids and sesquiterpenoids in the leaves of the hybrid between chrysanthemum and Artemisia vulgaris enhanced the resistance of the plant to aphids . In the present study, we obtained several DEGs related to terpenoid synthesis (Fig. 6A). There were three genes related to monoterpene synthesis, one basil synthase gene (Cluster-21353.197680), one myrcene synthase gene (Cluster-21353.180080) and three isoprenoid genes (Cluster-21353.97990, Cluster-21353.154989 and Cluster-21353.47033). All of these genes were strongly upregulated in B1 compared to A1, indicating their potential roles in the defense responses against aphids in the early stage after grafting on A. scoparia W. Furthermore, five genes involved in diterpenoid biosynthesis, including one gene (Cluster-21353.173831), were strongly upregulated in B1 compared to A1, and four genes (Cluster-21353.18772, Cluster-21353.318315, Cluster-21353.283531 and Cluster-21353.113455) were strongly upregulated in B2 compared to A2 and may play important roles in responding to aphids in the later stage of infestation.
The details of these genes discussed above are shown in Additional file 3: Table S2, which illustrates the involvement of secondary metabolites during aphid herbivory in Cm / As and Cm / Cm leaves, indicating that using the A. scoparia W. rootstock could alleviate aphid stress in chrysanthemums by altering these gene profiles, which play potential roles in defense responses to aphids.
Plant hormone signaling pathway involved in plant-aphid interaction
JA, SA and ET are three major phytohormones involved in the regulation of signaling networks related to aphid-infestation defense responses. As shown in Fig. 7A, hormone transduction pathway-related genes were expressed at different time points between Cm / As and Cm / Cm. A total of 21 DEGs were involved in several plant hormone signal transduction pathways, including cytokinins (CKs), ET, JA and SA, in this study. The details of these genes discussed above are shown in Additional file 4: Table S3.
The JA pathway and SA pathway are two main pathways involved in plant-induced defense . Phytohormonal crosstalk between JA- and SA-mediated signaling pathways is thought to underlie plant-mediated interactions among multiple insect species and the behavioral responses of parasitoids and predators [10, 42, 43]. The activation of SA signaling in response to aphid feeding [18, 44, 45] may suppress JA-dependent indirect defense responses. In our study (Fig. 7A), one LOX gene, which is a key enzyme in JA synthesis, was downregulated in B1 compared to B0, whereas the SABP genes, which are key enzymes in SA synthesis, were strongly upregulated in B1 compared to B0.
Exogenous SA treatment can enhance plant resistance to pathogenic bacteria and induce the expression of disease-related genes at the same time [46, 47]. SABP2 catalyzes the conversion of MeSA into SA, which is essential for inducing tobacco systemic acquired resistance (SAR) . In this study, three SABP2 genes (Cluster-21353.21806, Cluster-21353.149100 and Cluster-21353.197082) were upregulated by aphid infestation in B1 compared to A1 in the early stage. In addition, within 6 h of infestation with aphids, the CAT activity in the Cm / As and Cm / Cm plants decreased rapidly by 69.5% and 83.7%, respectively (Fig. 7B). This result might be due to the change in the conformation of CAT, which binds to the SA-binding protein (SABP1) , inhibiting the activity of CAT and activating the expression of disease course-related protein genes. We suggest that grafting chrysanthemum onto A. scoparia W. inhibited the activity of CAT through the upregulation of SABP2 genes after aphid infestation (Fig. 7C), and then the self-feedback mechanism was initiated, which amplified the signal transduction in the cell and ultimately induced the expression of the disease-related protein genes (Fig. 7G). Furthermore, in the SA-mediated SAR reaction of Arabidopsis, NPR1 is a crucial regulatory protein that impacts the SAR pathway downstream of the SA signaling pathway. During SAR, NPR1 induces PR gene expression, and even after induction by SA, BTH or INA, NPR1 expression levels increase [50, 51]. However, in this study (Fig. 7A), two NPR1 genes were downregulated in B1VSA1, suggesting that the NPR1 gene had different regulatory mechanisms on aphid stress after grafting onto A. scoparia W.
JA affects not only the growth of many plants but also the resistance of plants to pathogen infection. LOX and oxo-phytodienoic reductase (OPR) are not only key enzymes for JA synthesis but also important substances that induce defense mechanisms in plants [52-54]. As the receptor in the JA signaling pathway, COI1 (COR-insensitive1) can interact with SKP1 (S-phase kinase-associated protein1) . LOX genes were significantly upregulated by M. persicae feeding on A. thaliana leaves , M. nicotianae feeding on Nicotiana attenuata leaves , and M. euphorbiae feeding on tomato leaf tissues . In our study (Fig. 7E), the overall expression level of one LOX gene (Cluster-21353.151969) in the Cm / As plants was higher than that in the Cm / Cm plants at the same time point. Additionally, the activity of LOX (Fig. 7D) in Cm / As was significantly increased by aphid infestation and higher than that in Cm / Cm, which promoted the accumulation of JA. Compared with A2, B2 exhibited one OPR gene (Cluster-21353.142219) that was strongly upregulated. In addition, one COI1 gene (Cluster-21353.180331) and one MYC gene (Cluster-21353.197996) were strongly upregulated in B1 compared to A1 and responded rapidly to aphid stress in the early stage of infestation. In addition, protease inhibitors (PIs) are widely found in organisms, regulate many important biological activities in plants and have a protective effect against insects . These genes above all activated the expression of cytochrome P450 genes and protease inhibitor genes, which were downstream response genes (Fig. 7G) responding to aphid stress. The details of these genes discussed above are shown in Additional file 5: Table S4. In this study, four cytochrome P450 genes (Cluster-21353.342863, Cluster-21353.153679, Cluster-21353.345312 and Cluster-21353.344914) were upregulated by aphid infestation in B1 compared to A1. Protease inhibitors were divided into four categories, serine protease inhibitors, metal carboxypeptidase protease inhibitors, cysteine protease inhibitors and aspartic acid protease inhibitors, according to the active sites of the active enzymes . In our study, two cysteine protease inhibitor genes (Cluster-21353.197535 and Cluster-21353.189320) and one phospholipase A2 inhibitor gene (Cluster-21353.200786) were significantly upregulated by aphid infestation in B1 compared to A1. As important enzymes, the acetylcholine receptor inhibitor gene and the phospholipase A2 inhibitor gene can inhibit the nerve conduction of insects, which may play an important role in responding to aphids in the early stage of infestation after grafting onto A. scoparia W.
Some studies have proven that aphid infestation markedly increases the production of ET in the leaves of plants [60-62]. Ethylene-inducing xylanase (EIX) induces ET production and is an effective elicitor of plant defense responses in specific cultivated species of Nicotiana tabacum and Lycopersicon esculentum. In the present study, one EIX gene (Cluster-21353.186218) was strongly upregulated in B1 compared to A1, which may stimulate the accumulation of ET (Fig. 7F). CTR1 is a negative regulatory component downstream of the ET receptor and the first cloned gene in the ET signal pathway [63, 64]. In this study (Fig. 7A), one CTR gene (Cluster-21353.234433) was upregulated in B1 compared to A1 at an early time point after aphid infestation. Research shows that EIN2 has important effects in the ET signal pathway, and the loss of EIN2 gene function leaves the plant completely insensitive to ET . In this study (Fig. 7A), one EIN2 transcription factor gene was downregulated in B1 compared to B0, but it was highly expressed in A1. ET-responsive factor (ERF) encodes a transcriptional activator to promote several downstream ET-responsive genes. Additionally, MYC and ERF transcription factors, participate in the ET and JA signaling pathways and activate defense-related genes. In our study, nine ERF genes were involved; among them, four genes (Cluster-21353.197997, Cluster-21353.197998, Cluster-21353.176859 and Cluster-21353.148870) were strongly upregulated in B1 compared with A1 (Fig. 7A).
These DEGs were identified in the comparison of Cm / As and Cm / Cm, as discussed above, which illustrates the involvement of the plant hormone signaling pathway during aphid herbivory, indicating their potential roles and the complex connections in the defense responses against aphids after grafting chrysanthemum onto A. scoparia W.
Two layers of plant immunity are well defined: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) . As the first line of the innate immune response, PTI is triggered by the perception of herbivore-associated molecular patterns (HAMPs) in the case of herbivory and microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) by cognate plasma membrane-localized pattern recognition receptors (PRRs) in the case of microbial infection . The downstream immune signaling pathways are activated by different PRRs, including the receptor of kinases and the activation of various kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) . In this study, four genes in the MAPK signaling pathway were recognized (Fig. 8): one ROBH gene (Cluster-21353.196945) and one MAPK gene (Cluster-21353.172651), which were upregulated in B1 compared to A1, and one WRKY2209 gene (Cluster-21353.216549) and one ROBH gene (Cluster-21353.177651), which were upregulated in B2 compared to A2.
Ca2+ regulates many important physiological processes. Transcriptome and metabolome changes in Arabidopsis were surveyed at 6 h, 12 h, 24 h and 48 h after B. brassicae infestation, revealing that reactive calcium is involved in early signaling . Plant defenses against phloem-feeding insects involve multiple signaling cascades, and molecular genetic studies on the model plant A. thaliana have demonstrated that leucine-rich repeat (LRR) family receptor-like kinases and calcium signaling proteins are partially activated by phloem feeding . MHCII molecules can mediate the transmission of reverse signals and influence and regulate many physiological processes in immune cells . Studies have shown that CDPK2 and MHCII can regulate each other in two major antigen presenting cells . In the present study (Fig. 8), four CDPK genes (Cluster-21353.147746, Cluster-21353.90102, Cluster-21353.147746 and Cluster-21353.90102) and three MHCII genes (Cluster-21353.251721, Cluster-21353.196511 and Cluster-21353.177618) were specifically upregulated in B1 compared to A1, which may play an important role in the early stage of responding to M. sanbourni infestation in Cm / As seedlings.
Two cloned aphid resistance genes, Mi-1.2, confer resistance to the potato aphid, Macrosiphum euphorbiae (Thomas) [72, 73], and Vat mediates resistance to the cotton aphid Aphis gossypii Glover [74, 75], all of which belong to the NBS-LRR family. Similarly, we found four such DEGs (Cluster-21353.336850, Cluster-21353.79718, Cluster-21353.149283 and Cluster-21353.102248) between the Cm / Cm plants and the Cm / As plants, but these DEGs require further cloning and functional identification to confirm the presence of the NBS-LRR region.
Furthermore, in our study (Fig. 8), there were many DEGs related to tyrosine protein kinases and serine/threonine protein kinases in the interaction between the plants of the Cm / Cm and the Cm / As. Eight PAK genes and six PTK genes were specifically upregulated in B1 compared to A1. These protein kinase genes may be closely related to grafting to improve the aphid resistance of C. morifolium T. (The details of these genes discussed above are shown in Additional file 6: Table S5)