Elevated production of reactive oxygen species is related to host plant resistance to sugarcane aphid in sorghum.

ABSTRACT

Sugarcane aphid (Melanaphis sacchari) is a phloem-feeding insect that severely affects the growth and productivity of sorghum and other related crops. While a growing body of knowledge is accumulating regarding plant, and insect interactions, the role of reactive oxygen species (ROS) against aphid infestation in sorghum has not been established yet. Here, the involvement of H₂O₂ and ROS detoxification enzymes in host plant resistance to sugarcane aphid in sorghum was demonstrated. The H₂O₂ accumulation and expression patterns of selected ROS scavenging enzymes including ascorbate peroxidase (APX), glutathione S transferase (GST), superoxide dismutase (SOD), and catalase (CAT) in response to sugarcane aphid infestation at 3, 6, 9, and 12 days post infestation (dpi) in resistant (Tx2783) and susceptible (Tx7000) sorghum genotypes were assessed, respectively. A significant increase in H₂O₂ accumulation was observed in resistant genotypes at all time points studied as compared to susceptible plants. Furthermore, gene expression analysis revealed that in responding to attack by sugarcane aphid, antioxidant genes were induced in both genotypes, but much stronger in the resistant line. Furthermore, aphid survival and fecundity were significantly inhibited in resistant plants compared to susceptible plants. Taken together, our results suggest that the elevated accumulation of H₂O₂ and the strong upregulation of the antioxidant genes in sorghum may have contributed to host plant resistance in Tx2783 against sugarcane aphid but the weak expression of those antioxidant genes in Tx7000 resulted in the failure of attempting defense against sugarcane aphid. This report also provides the experimental evidence for the role of ROS involvement in the early defensive response to an attack by sugarcane aphid in sorghum.

Introduction

Sugarcane aphid (Melanaphis sacchari) is a major pest of sorghum [Sorghum bicolor (L) Moench] in south Asia, Africa, central, and south America¹. It was first reported in sugarcane in the United States in Florida,² and later in Louisiana.³ Since then, sugarcane aphid (SCA) was considered a sporadic pest of sugarcane until recently, when a sudden outbreak in sorghum occurred in Beaumont, Texas in 2013. Now, SCA is considered a major pest of sorghum and has seriously threatened sorghum production in more than 20 states in the US.⁴ SCA can be quickly dispersed by wind and agriculture machinery, which makes it difficult in management of sugarcane aphid. SCA mainly feeds and colonizes on the underside of the lower leaves and main stem (in younger plants); however, during the outbreak the aphid attack sorghum plants at all developmental stages and infest various parts of sorghum including leaf, stem, panicle, and grains, causing severe yield loss and quality of grain.^1,5 SCA takes nutrition from its host by piercing plant tissues with its stylets and sucking the phloem sap, causing loss of nutrition in the plant and reduction in plant growth.¹ Moreover, SCA produces honeydew in SCA-infested leaves and panicles, which promotes the development of sooty mold on plants, which ultimately reduces photosynthetic area and efficiency, and inhibits panicles/grains development as well as affects mechanical harvesting due to clogging of grains.

Sorghum, an important cereal crop in the United States for food, feeder, and biofuel industry with approximately 2 USD billion economic impacts in 2015, but its production has been declining in recent years by 21.6% and 19.5% in 2015/2016 and 2016/2017, respectively, where the epidemic SCA was part of the reasons.⁶ To date, various genetic sources of resistance to SCA were reported but mechanisms underlying the host resistance remain to be elucidated. Also, while a growing body of knowledge is accumulating regarding the sorghum-sugarcane aphid pathosystem, less is known about host plant response to SCA infestation in sorghum. Herein, we aimed to investigate the reactive oxygen species (ROS) mediated host responses to sugarcane aphid in resistant (Tx2783) and susceptible (Tx7000) lines to better understand the defense role of H₂O_2, a most stable form of ROS in plants.

Reactive oxygen species (ROS) are a highly reactive form of oxygen and play pivotal roles in the regulation of a large array of cellular processes, such as growth and development as well as response to biotic and abiotic stresses.^7–11 ROS is an unavoidable natural by-product of various aerobic metabolic processes, such as photosynthesis, glycolysis, or oxidative phosphorylation in plant and animal cells. The major forms of ROS in plant cells are superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^•)^12,13 which are mainly produced in apoplast, chloroplast, mitochondria, and peroxisomes. Production of a high level of ROS in a cell causes oxidative damage to membranes (lipid peroxidation), pigments, proteins, and nucleic acids, leading to cause cell death eventually, which is known as an oxidative stress.^14,15 To cope with the detrimental effect of ROS, plants developed an antioxidant mechanism to protect the plant from oxidative damage by removing excess ROS from the cell. The antioxidant system includes ROS detoxifying proteins (e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidases (GPX), glutathione S-transferase (GST), peroxiredoxin (PRX)), antioxidant (e.g., ascorbate, glutathione, and nicotinamide adenine dinucleotide phosphate (NADPH)), and free transient metals (e.g., Fe²⁺).^16–21 ROS are known to serve as a signaling molecule involved in the regulation of cellular growth, stomatal closing,²² programmed cell death,¹⁵ response to abiotic^15,23 and biotic stresses.^{16,17,19,22,24,25} Recent studies showed that heavy metals (Pb and Cd) and saline-alkali stress (NaCl and NaHCO3) can also modulates ROS and anti-oxidant enzymes in plants.^26,27 Increasing evidence demonstrated that rapid accumulation of ROS takes place at the site that is attacked by plant pathogen or pest, which is known as oxidative burst,²⁸ causes the death of infected host cell to prevent further spread of the pathogen.^15,29 This phenomenon is called the hypersensitive response (HR). Furthermore, enhanced accumulation of ROS can also activate SA-mediated defense response in host plants.^30–33

Materials and methods

Plant growth and aphid culture conditions

In this study we used susceptible (Tx7000) and resistant (Tx2783) lines of sorghum. The Tx2783 line was known as a source of resistance to greenbug biotype C and E³⁴ and was also found resistant to sugarcane aphid.³⁵ The Tx7000 line was an elite variety used in the US. during the 1940s-50s prior to the development of hybrids³⁶ and has been used as a susceptible check³⁷ in SCA infestation experiments. Seeds from Tx2783 and Tx7000 sorghum (Sorghum bicolor) lines were treated with a fungicide (Captan 50 W, Bonham, TX) and planted separately in a plastic pot (7.5 cm in diameter, 7.1 cm in height) filled with potting compost (Sungro Professional Growing Mix, Agawam, MA). Each pot was covered with clear plastic cages (4.3 cm in diameter and 55 cm in length) and kept in a tray filled with water. Seedlings were grown in a greenhouse for 8–10 days (i.e., at the 3-leaf stage) at constant temperature (28 ± 2°C) and 60% relative humidity under constant photoperiod of 14 h-light/10 h-dark. Sugarcane aphid colonies were cultured on seedlings of susceptible sorghum line (Tx7000) in pots (15 cm x 14 cm) fitted with 12.5 cm x 30 cm cylinders of Lexan^TM (SABIC Polymershapes, Tulsa, OK) sleeve cages with netting cloth covering on the top.

Aphid infestation and counting

Sorghum seedlings at the three-leaf stage were infested with 20 adults apterous sugarcane aphid to the adaxial surface of the first leaf with paintbrush. Only healthy seedlings with similar height were included in the experiments. To restrict aphid scape and avoid infestation from unwanted pests/pathogens, each infested plant was covered with a transparent cylindrical cage with nylon mesh on the top. The number of aphids per plant was counted in both sorghum lines at 3, 6, 9, and 12 days post infestation (dpi). Control plants were also covered in cages but not infested with aphids.

H₂O₂ measurement

To visualize H₂O₂ accumulation in sugarcane aphid infested and control leaves, the lower leaf was collected and stained with 3,3ʹ-diaminobenzidine (DAB) (Sigma–Aldrich, St. Louis, MO, USA) as described by Shao et al.³⁸ with some modifications. In brief, three leaves from three independent biological replicates were collected at 3, 6, 9, and 12 dpi and aphids were gently brushed away from infested leaves. The leaves were then immersed in water with Triton-X-100 (0.01%) and DAB solution (1 mg/ml) with adjusted pH 3.8 and incubated overnight with gentle agitation at room temperature in dark. After incubation, leaves were bleached in alcoholic lactophenol (2:1:1, 95% ethanol: lactic acid: phenol) at 65°C for 30 min, rinsed with 50% ethanol, and then rinsed with sterile water. Stained leaves were photographed with a phone camera, and then observed under a stereomicroscope system (IX71, Olympus Co., Tokyo, Japan) and photographed with an affiliated CCD camera. The experiment was repeated for 3 times.

The quantification of H₂O₂ was determined by chemiluminescence assay as described previously³⁹ with few modifications. In brief, at least five leaves were collected from five independent plants (one per plant) at 3, 6, 9, and 12 dpi from aphid infested and control plants for each genotype. Each leaf was excised into five-leaf discs (4 mm diameter) which were incubated overnight in sterile water in a 96-well assay plate (Corning Incorporated, Kennebunk, ME) in dark at room temperature to eliminate wounding response. The water from the well was replaced with 100 μl solution containing 50 μM of luminol (Sigma, catalog #A8511-5 G) and 10 μg/ml of horseradish peroxidase (HRP; Sigma, catalog # P6782) in 200 mM KOH. Immediately, ROS measurement was performed with SynergyH1 plate reader (Biotek) for a period of 60 min. The values of H₂O₂ production from each genotype were represented as relative light units (RLUs).

Assessment of plant phenotype

Following infestation, plant height, number of aphids, and plant damage rating (chlorosis/necrosis) was recorded in resistant and susceptible genotypes, respectively, at 1, 2, 3, 6, 9, 12, and 14 dpi. At least 10 independent plants were measured, and the experiments were repeated 3 times. Each plant was given a damage score from 1 to 6 based on the percent of damage in the plant (Table 1). A damage score 0 corresponds to plant with no damage, 1 to plant with <20% damage, 2 to 21–40% damage, 3 to 41–60% damage, 4 to 61–80% damage, 5 to >80% damage, and 6 to dead plant.

Table 1.

Plant height differences (cm), number of aphids, and damage ratings for two sorghum lines subjected to sugarcane aphid infestation

	Plant height difference		Aphid Count		Damage ratings
DPI	Tx7000	Tx2783	Tx7000	Tx2783	Tx7000	Tx2783
0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0	0
1	0.75 ± 0.75	1.15 ± 0.35	24 ± 1.2	21.3 ± 1.2	0	0
2	2.25 ± 0.75	1.25 ± 0.55	46 ± 3.6	39 ± 2.7	1	0
3	2.5 ± 0.5	0.95 ± 0.65	60 ± 6.0	51 ± 2.1	1	0
6	4.9 ± 0.8	3.25 ± 0.75	222 ± 20.6	106 ± 10.9	1	0
9	7.25 ± 1.75	3.85 ± 1.75	834 ± 26.1	310 ± 23.4	2	0
12	10 ± 2	2.66 ± 0.35	1146 ± 27.7	541 ± 30.2	5	1

RNA isolation and quantitative real-time (qRT)-PCR

Total RNA was extracted from aerial parts of sorghum plants using TRIzol reagent (Invitrogen) following the manufacturer’s instructions and then treated with DNase (Turbo DNA-free kit, Thermofisher, Waltham, MA) for 30 min at 37°C. The purified RNA was reversely transcribed using GoScript reverse transcriptase kit (Promega, Madison, WI) and the resultant cDNA was diluted to five-fold with sterile water before used for the qRT-PCR reaction. Primers were designed using the IDT DNA program (https://www.idtdna.com/PrimerQuest/Home/Index), which are listed in supplementary Table S1. A sorghum β-tubulin gene (Sobic.002g350400) was used as the internal control as described previously.⁴⁰ Quantitative real-time PCR (RT-qPCR) was performed on a Bio-Rad icycler thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the iTaq™ universal SYBR® green supermix (Bio-Rad Laboratories, Inc.). The qRT-PCR reaction was performed in a total volume of 10 μl, containing 1 μl of diluted cDNA, 0.4 μl (10 μM) of reverse and forward primers, 5 μl of SYBR green master mix, and 3.2 μl of ddH2O using the following conditions: one cycle at 95°C for 3 min, 40 cycles at 95°C for 10 s, and 55°C for the 30 s, followed by one cycle each of 1 min at 95°C and 55°C. The final melting curve was of 81 cycles at 55°C for the 30 s. The relative expression level of each gene was calculated using the 2− ΔΔCt method⁴¹ and the data were the averages of three biological and two technical replicates.

Results and discussion

Infestation of sugarcane aphid induces H₂O₂ in resistant sorghum

The effect of aphid infestation on H₂O₂ production was investigated in resistant (Tx2783) and susceptible (Tx7000) plants. The level of H₂O₂ in susceptible and resistant sorghum lines was measured during the co-culture of sugarcane aphid and plant. For qualitative estimation of H₂O₂ using DAB assay, the lowermost leaves were collected from healthy and aphid-infested plants, respectively, at 3, 6, 9, and 12 dpi, and stained in DAB solution. H₂O₂ accumulation is consistently higher in aphid-infested resistant genotype compared to susceptible plants at all time points as indicated by more intense dark reddish-brown color (Figure 1). The observation suggests that the resistant genotype produces more H₂O₂ in response to sugarcane aphid infestation. However, ROS level is not much changed in aphid feeding susceptible genotype except 9 dpi where a significant increase in H₂O₂ accumulation was observed. Furthermore, we performed a chemiluminescence assay to validate the results obtained from the DAB assay. The chemiluminescence data showed that H₂O₂ level increased at all examined time points in Tx2783 plants upon sugarcane aphid infestation (Figure 2), confirming our DAB assays results. Noticeably, H₂O₂ accumulation was not occurred in aphid feeding Tx7000 plants in comparison to healthy plants at all time point except 9 dpi (Figure 2), mimicking our DAB assay results. It could be possible that the H₂O₂ accumulation triggered in Tx7000 by massive colonization of aphids in leaves at 9 dpi which causes physical damage to epidermal and mesophyll cells (Figure 3). At the beginning of an infestation, sugarcane aphids probed available plants to select suitable sources of feeding during their initial interaction; then once aphids settled down on the host plants, they started to colonize on the host. As aphid feeding proceeded, necrotic phenotype became prominent at the same time (9 dpi) as H₂O₂ accumulation took place in susceptible genotype, suggesting ROS induced cell death and necrosis in susceptible genotype. Moreover, the intensity of DAB staining is relatively less in aphid-infested Tx7000 plants when compared to Tx2783 (Figure 1) at 9 dpi, suggesting their inefficiency in stopping aphids feeding. At 12 dpi, SCA-infested susceptible plants exhibited suppressed H₂O₂ level when compared to uninfested plants. Taken together, these results suggest that resistance to SCA infestation in Tx2783 is associated with consistent-enhanced accumulation of H₂O₂ in sorghum leaves.

Figure 1.

Accumulation of H₂O₂ in sorghum leaves as determined by DAB staining at 3 (a), 6 (b), 9 (c), and 12 (d) days following sugarcane aphid infestation. The dark reddish-brown staining represents the accumulation of H₂O₂. Upper panel represents images of a leaf and lower panel represents corresponding images as seen under stereomicroscope

Figure 2.

Accumulation of H₂O₂ in sorghum leaves as determined by chemiluminescence assay at 3 (a), 6 (b), 9 (c), and 12 (d) days following sugarcane aphid infestation. RLU, relative light units

Figure 3.

Mean number of aphids in resistant (blue) and susceptible (green) sorghum lines at 1, 2, 3, 6, 9 and 12 dpi

The uplift accumulation of ROS was reported as the earliest defense event triggered by various aphid infestations, including sugarcane aphid, in cereal crops.^9,38,42–45 It has been proposed that ROS accumulation could induce aphid resistance and ROS suppression results in susceptibility to aphid in plants.^46,47 The infestation of plants by aphid allows injection of saliva into phloem cells, containing various effector proteins, hydrolytic enzymes, and toxic compounds which trigger plants to perceive aphid invasion and may stimulate ROS accumulation.⁴⁸ A recent study reported that a cathepsin-type salivary protease secreted by Myzus persicae binds to tobacco cytoplasmic kinase ENHANCED DISEASE RESISTANCE 1-like (EDR1-like), which activates ROS accumulation in tobacco phloem, thereby suppressing their colonization in the host plant.⁴⁹ Prolong accumulation of ROS can lead to peroxidation of lipids and chloroplast, and cause damage to macromolecules results in programmed cell death.^14,15,50 Thus, it is crucial for the survival of plants to effectively scavenge excess ROS. Several researches demonstrated that resistant genotypes induce ROS accumulation and activate ROS scavenging mechanism (enzymatic or/and non-enzymatic) to cope with the deleterious effect of ROS accumulation and deter invading aphids. For instance, we have previously reported that the enhanced expression of peroxidase in greenbug (Schizaphis graminum) feeding resistant sorghum genotype⁵¹ which induced ROS production. Russian wheat aphid infestation induced H₂O₂ accumulation in wheat compared to near-isogenic plants.⁴⁸ Similarly, elevated accumulation of H₂O₂ has been reported from an aphid-resistant cultivar of sorghum (HN16),³⁸ and pepper (NL population).⁵² Similarly, ROS accumulation was reported in resistant sorghum genotype (SC146) upon Colletotrichum sublineolum infection, whereas ROS was not accumulated in susceptible genotype (BTx623).⁵³

Resistant genotype inhibits survival and fecundity of sugarcane aphids

To gain an understanding of the effect of aphid infestation on plant growth, aphid survival, and fecundity in susceptible and resistant genotypes, we performed an experiment to assess the number of aphids, plant height, and plant damage at selected time points (1, 2, 3, 4, 6, 9, and 12 dpi). SCA fecundity was determined by placing 20 sugarcane aphids of the same size at the base of the first leaf, and the number of aphids was counted on each plant (n = 10) at the above-mentioned time points. SCA fecundity was found significantly diminished on Tx2783 plants compared to Tx7000, with an average of 1146 ± 27.7 aphids per plant whereas the average number of aphids was 541 ± 30.2 in Tx2783 at 12 dpi (Figures 3 & 4, Table 1). Most susceptible seedlings were completely wilted at 12 dpi and dead at 14 dpi (Figure 4). Therefore, we were not able to collect data from Tx7000 plants after 14 dpi, and the experiment was stopped at that point. The sharp decline of aphid numbers in Tx7000 lines at 14 dpi was due to the death of the plants (Figure 3, Table 1). Based on damage rating, Tx2783 showed a strong resistance with an average damage score being 1 or less, whereas Tx7000 exhibited the highest level of susceptibility with a rating of five, the highest average damage score (Figure 3, Table 1). Damage scores three and below is considered highly resistant, whereas scores five and six are considered highly susceptible. The sugarcane aphid colonization reached all over the plants in Tx7000 lines as early as 2 dpi and start showing chlorosis and necrosis at 3 and 4 dpi, respectively. Whereas sugarcane aphid is rarely colonized in upper leaves in Tx2783 seedlings and shows chlorosis and necrosis symptoms at 12 dpi (Table 1). Plant height differences between control and aphid-infested plants were significantly affected in susceptible genotype upon sugarcane aphid infestation (Table 1). Whereas no significant differences in plant height were reported in the resistant genotype. More than 10 cm difference in height was measured in Tx7000 control vs Tx7000 infested plants. In contrast, the maximum height difference between control and sugarcane aphid-infested Tx2783 plants is 2.66 cm (Table 1), which was not statistically significant. These findings are in line with previous studies^37,54,55 in which Tx2783 also exhibits an enhanced level of tolerance and antibiosis.

Figure 4.

Sorghum plants infested with sugarcane aphid (SCA). Upper panel: Sorghum susceptible (Tx7000) and resistant (Tx2783) genotypes after various time points of SCA infestation. Lower panel: enlarged view of infested stem of respective sorghum genotypes

Antioxidant genes strongly induced in resistant genotype in response to feeding by sugarcane aphid

DAB and chemiluminescence assays (Figures 1 & 2) revealed H₂O₂ modulation by sugarcane aphid infestation in two studied sorghum lines. The antioxidant capacity of plants is important to cope with the detrimental effect ROS generated in response to aphid infestation. Here we aimed to investigate comparative molecular responses to ROS accumulation triggered by aphid infestation in resistant and susceptible genotypes. To elucidate the role of genes involved in ROS detoxification in resistant and susceptible sorghum genotypes during sugarcane aphid infestation, relative expression of genes coding for selected ROS scavenge enzymes in foliar tissues of the aphid-infested and non-infested leaves was estimated using qRT-PCR. At least one gene was selected from each following enzyme families; superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST), for expression analysis.

Superoxide dismutase (SOD)

The expression of two tested genes (SOD1 and SOD2) encoding for SOD was upregulated upon sugarcane aphid infestation when compared to non-infested seedlings in both genotypes at 3, 6, 9, and 12 dpi (Figure 5). However, the expression of SOD1 and SOD2 was significantly higher in resistant genotype when compared to susceptible genotype at all time-points except 12 dpi. The transcript levels corresponding to SOD1 responded quickly in resistant sorghum seedlings, reaching a maximum of over 13-fold compared to control whereas SOD2 transcript level was reached its highest level (5.1-fold) at 6 dpi, and slowly returning to their lowest level (about two-fold) by 12 dpi. A moderate increase in transcript levels was detected in susceptible plants upon sugarcane aphid infestation with a 2.3-fold increase of SOD2 and 1.9-fold increase of SOD1 at 3 and 12 dpi, respectively. An elevated level of SODs provides the first line of defense against oxidative damage by dismutation of superoxide to molecular oxygen and H₂O₂. Furthermore, we observed a higher level of H₂O₂ accumulation in resistant plants as compared to susceptible plants under aphid infestation (Figures 1 & 2) which might be associated with higher SODs activity in the resistant line. The increase in SODs transcript in the sorghum seedlings upon sugarcane aphid infestation might be a defensive response and suggested its role in combating elevated ROS levels triggered by aphid infestation. An increase in transcript abundance of SODs has been reported in many crops and model plant species upon aphid/pest infestation. Sytykiewicz (2014) reported increased expression of three maize SODs (SOD1, SOD3.4, and SOD9) in relatively resistant maize cultivar (Ambrozja) infested with grain aphid or bird cherry-oat aphid. Overproduction of SOD in cassava resulted into resistance to mites.⁵⁶ Similarly, increased activity of SOD enzymes reported in cucumber against Bemisia tabaci,⁵⁷ in pea in response to pea aphid,⁵⁸ in rose to aphid infestation,⁵⁹ and in wheat against greenbug infestation.⁶⁰ The slight increase in SOD expression in Tx7000 may be a failed attempt to detoxify the ROS induced by aphid-infestation. These reports are congruent with our results suggesting susceptible and resistant genotype differs in their transcriptional response of SOD to aphid herbivory and involved in aphid resistance in sorghum.

Figure 5.

Effect of SCA infestation on expression of antioxidant genes in Tx7000 and Tx2783 lines. Quantitative expression of superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST), and ascorbate peroxidase (APX) in sorghum at 3 (a), 6 (b), 9 (c) and 12 (d) dpi in response to sugarcane aphid infestation

Catalase (CAT)

Changes in transcript abundance of CAT1 and CAT2 genes were observed in both genotypes exposed to sugarcane aphids. However, a much stronger increase in CAT genes at the mRNA level was detected in the resistant line in each time point studied (Figure 5). Expression of CAT1 and CAT2 genes peaked to 9.2-fold and 18.7-fold above the control, respectively, at 3 dpi in Tx2783, followed by a slow decrease in its accumulation at later stages of the aphid infestation. In Tx7000 seedlings, the highest aphid-provoked increase in expression of CAT1 and CAT2 transcripts was demonstrated at 12 dpi (2.8-fold) and 3 dpi (8-fold), respectively. Catalase is one of the important ROS scavenging enzymes which effectively inhibit H₂O₂ by converting it to water and oxygen and play a significant role in plant defense.¹⁴ We found a strong induction of catalase transcripts and a significant reduction in aphid counts in the resistant line, which suggests its role in plant defense against the sugarcane aphid. Expression of catalase was also found increased in aphid-infested Tx7000 when compared to non-infested control, but the level of catalase was lower in Tx7000 compared to Tx2783. However, increased catalase levels in susceptible plants could not provide resistance to sugarcane aphids which could be due to the catalase expression could not meet the threshold level to inhibit aphid reproduction and survival. These findings are in line with previous studies that demonstrated higher catalase accumulation in aphid resistance plants and are effective in providing resistance to aphids and pests. For instance, increased catalase activities were found in resistant alfalafa lines against spotted alfalfa aphid,⁶¹ in cassava to mites (Tetranychus cinnabarinus),⁵⁶ in cucumber seedlings to Bemisia tabaci,⁵⁷ and in tobacco to Bemisia tabaci⁶² and Myzus persicae.⁶³ Furthermore, ROS has been shown to induce SA accumulation and thereby activates local and systemic defense against pathogens via pathogenesis-related (PR) protein expression.^30–33 A few available reports stated a positive correlation between the expression of catalase and SA-mediated defense response.^33,62 The increased catalase transcripts in resistant sorghum line upon aphid feeding may be contributed by the elevated accumulation of salicylic acid (SA) indicating SA-mediated defense response in Tx2783 against the sugarcane aphid.

Ascorbate peroxidase (APX)

The upregulation of APX1 was observed in Tx2783 plants exposed to sugarcane aphid infestation (Figure 5) at all studied time points. The abundance of APX1 was induced by 7.4-fold in resistance plants compared to control at 3 dpi, followed by a decrease to 2.9-fold at 6 dpi and again increased to 8.6-fold and 4-fold at 9 and 12 dpi, respectively. In susceptible plants, its transcript was slightly increased to 1.8-fold and 2.1-fold compared to control plants upon sugarcane aphid infestation at 3 dpi and 12 dpi, respectively, and then the transcripts decreased to the level of control at 6 and 9 dpi. APX is an important enzyme of the ascorbate-glutathione (AsA-GSH) cycle where APX catalyzes the reduction of H₂O₂ to monodehydroascorbate radical (MDHA) and water.¹⁴ Several studies reported that ascorbate peroxidase responds to biotic stresses in multiple crops. Transcriptional analysis in switchgrass during greenbug aphid infestation showed differential expression of the ascorbate peroxidase genes. Six of 12 studied ascorbate peroxidase genes were induced in response to greenbug aphid infestation in switchgrass.⁶⁴ APX was higher in resistant cultivars and lower in susceptible cultivars of rose exposed to aphids.⁵⁹ Sytykiewicz (2014) found an elevated level of transcripts of six APX genes (APX1, APX2, APX4, APX5 APX6, and APX7) in resistant maize genotype as compared to susceptible plants upon bird cherry-oat aphid or grain aphid infestations. Recently, it has been reported that sorghum APX1 (SbAPX1) plays an important role in RMES1-mediated resistance to sugarcane aphids in sorghum.³⁸ Similarly, induced transcriptional responses of peroxidases including APX1 have been documented in sorghum in response to sugarcane aphid infestation in resistance genotype⁶⁵ and in wheat upon greenbug infestation.⁶⁰

Glutathione S- transferase (GST)

Higher differential upregulation of both GST genes (GST1 and GST3) was observed in Tx2783 genotypes in response to infestation with sugarcane aphid when compared to susceptible seedlings (Figure 5). In the resistance genotype, the maximum increase in transcript abundance was reported for GST1 (81.6-fold) and GST3 (5.1-fold) at 9 dpi and 12 dpi, respectively. A weak induction of GST expression was observed in susceptible genotypes. Sugarcane aphid-evoked enhancement of GST1 and GST3 transcripts were noted (1.3- and 2.2-fold, respectively) at 12 dpi in susceptible plants (Figure 5). Overall, a higher increase in expression of GSTs occurred in resistant seedlings as compared to susceptible genotypes, suggesting the defense role of GST against sugarcane aphids in sorghum.

Plant GSTs are a family of enzymes which primarily catalyzes glutathione-mediated detoxification reactions of endogenous substrates and xenobiotics to less toxic compounds with greater solubility in water, improving their sequestration in vacuoles.⁶⁶ Furthermore, GSTs are involved in the transport of flavonoids, programmed cell death, signal transduction pathways, and scavenging oxidative stress.^67,68 GST encompasses about 2% of the total foliar proteins in cereal crops⁶⁹(Dixon et al. 2002). Limited studies have been reported on sorghum GST response to phloem-feeding aphids. Zhu-Salzman⁴² elucidated that enhanced expression of four GST genes (GST1, GST2, GST3, and GST4) in sorghum when attacked by greenbug aphid after 48 h of infestation. It has been demonstrated that several genes involved in the glutathione metabolism pathway, including GST, were up-regulated mostly in resistant sorghum genotype in response to sugarcane aphid infestation at 14 dpi at the transcriptome level.⁶⁵ Recently, Shao et al.³⁸ reported differential expression of five sorghum GST genes (GST1, GST2, GST3, GST4, and GST5) when exposed to sugarcane aphid in sorghum. Besides, GST expression has been reported upon virus (Sugarcane mosaic virus)⁷⁰ and fungal (Colletotrichum sublineolum)⁷¹ infection in resistance sorghum genotypes. In this study, we demonstrated that there is considerable temporal and genotype-dependent variation in transcriptional response in GST1 and GST3 genes. Strong induction GSTs expression in resistant line may be attributed as its role in aphid defense. The defense role of GST against aphids has been reported from other plant–aphid interactions. In maize, 9 GST genes (i.e., GST1, GST9, GST11, GST16, GST18, GST23, GST24, GST31, and GST38) were reported enhanced gene expression and enzyme activity in response to bird cherry-oat aphid and grain aphid.^43,44 Induced expression of GST genes during incompatible plant–microbe interaction has been also demonstrated in rice to Rice tungro spherical virus⁷² and to Xanthomonas oryzae pv. oryzae,⁷³ in wheat to Puccinia recondite f. sp. tritici,⁷⁴ and in barley to Fusarium graminearum.⁷⁵ These studies suggest that GST is induced in response to various plant-pathogen/pest interactions including aphids and plays a crucial role in host defense response in crops.

Taken together, this study showed that H₂O_2, an uncharged and more stable ROS member, plays a central role in response to aphid infestation by H₂O₂-mediated regulation of antioxidant gene expression and signal transduction cascades, as summarized in Figure 6. We found steady-state induction of H₂O₂ in resistant line upon sugarcane aphid infestation, whereas H₂O₂ level was suppressed at all the time points studied except 9 dpi in susceptible genotype. The induced H₂O₂ level served as a signal molecule to activate various defense pathways such as ROS scavenging proteins, MAPK, CDPK, and SA-mediated defense pathways, which led to host resistance to sugarcane aphid in Tx2783. In contrast, suppress H₂O₂ levels in susceptible lines failed to activate defense pathways at a minimum threshold levels to combat aphid infestation, leading to susceptibility in Tx7000. We have limited our scope of this study to only selected genes associated with the ROS scavenging pathways in this study, additional experiments are needed to include other enzymes and remaining members of the enzyme families to get a better understanding of the molecular mechanisms of ROS-mediated aphid resistance in sorghum. Furthermore, interactions of H₂O₂ with other plant defense pathways, including plant hormones, transcription factors should be elucidated in detail to fill the gap in the current knowledge of the genetic and metabolic basis of aphid resistance in sorghum.

Figure 6.

Schematic overview of sorghum-sugarcane aphid interactions: A proposed model for ROS-mediated defense signaling pathways in sorghum in response to sugarcane aphid infestation

Conclusion

Sorghum-sugarcane aphid interaction is a relatively less explored area of research in the field of cereal–aphid interactions. To the best of our knowledge, this is the first comparative study to document changes in ROS homeostats and expression of major antioxidant genes, along with phenotypic changes following sugarcane aphid infestation in resistant (Tx2783) and susceptible (Tx7000) lines. Qualitative and quantitative estimation of ROS in aphid-infested leaves showed that rapid accumulation of ROS occurred in the resistant line compared to the susceptible line upon exposure to sugarcane aphids. Gene expression analysis revealed that the studied genes were induced in both genotypes upon aphid infestation; however, strong induction was observed in the resistant line at all the time points. These results imply that the existence of ROS scavenging systems is critical to maintain ROS homeostasis; thus, providing host plant defense against invading aphid in sorghum. This study also revealed that different members of the same gene family or same genes over different time points with varied gene expression patterns upon aphid infestation; thus, underscoring their specific defense roles to against aphid infestation. Finally, H₂O₂ accumulation and enhanced expression of ROS detoxifying enzymes contributed to sugarcane aphid resistance in sorghum.

Funding Statement

This work was partly supported by funding from USDA-ARS, and additional funding from USDA-ARS, CRIS project, Grant Number: [3072-21000-009-00D (YH)].

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.