Enhanced aphid detoxification when confronted by a host with elevated ROS production

Abstract

Reactive oxygen species (ROS) plays an important role in plant defense responses against bacteria, fungi and insect pests. Most recently, we have demonstrated that loss of Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) function releases its suppression of aphid-induced H₂O₂ production and cell death, rendering the bik1 mutant more resistant to green peach aphid (Myzus persicae) than wild-type plants. However, little is known regarding how ROS-related gene expression is correlated with bik1-mediated resistance to aphids, or whether these aphids biochemically respond to the oxidative stress. Here, we show that the bik1 mutant exhibited elevated basal expression of ROS-generating and -responsive genes, but not ROS-metabolizing genes. Conversely, we detected enhanced detoxification enzymatic activities in aphids reared on bik1 plants compared to those on wild-type plants, suggesting that aphids counter the oxidative stress associated with bik1 through elevated metabolic resistance.

Constantly challenged by pathogen infection and insect herbivory, plants have developed a broad spectrum of defense mechanisms, ranging from physical barriers to chemical traits, to counter the biotic stresses.^1,2 In parallel, insects are perpetually evolving adaptive strategies behaviorally, structurally, physiologically and biochemically to mitigate the impact of host plant defense.³ Aphids use their highly modified mouthpart to ingest nutrients from phloem tissue. Such a specialized feeding style causes very limited tissue damage, thus avoiding strong wound-induced plant defense responses often provoked by chewing insects. Furthermore, salivary secretions inhibit sieve tube occlusion to ensure continued nutrient flow in phloem tissue and aphid stylets.^4-7 Nevertheless, the deceptive feeding can still trigger responses in host plants.

Reaction oxygen species (ROS) are strongly associated with plant defense responses. In plant-microbe interactions, local production of ROS is one of the earliest cellular responses following pathogen recognition, preceding the hypersensitive response (HR). This subsequent rapid and localized cell death effectively contains pathogens at the site of entry and triggers systemic defense response.^8-10 Herbivorous insects, in particular the phloem sap-feeding aphids, also induce HR-like cell death in host plants.^11,12 Relative to pathogens, however, much less is known regarding the role of the HR in defense against aphids. Russian wheat aphid (Diuraphis noxia)-resistant barley (Hordeum vulgare) exhibits a stronger HR than the susceptible cultivar.¹³ Resistance conferred by Vat, a member of the R gene family, is associated with fast microscopic HR specific against Aphid gossiypii.¹⁴ However, aphid-induced H₂O₂ production and HR confers resistance only to the bluegreen aphid (Acyrthosiphon kondoi), but not to the pea aphid (Acyrthosiphon pisum), suggesting that cell death and resistance can be uncoupled.^15,16 In Arabidopsis thaliana, hypersenescence mutants that undergo spontaneous cell death show enhanced resistance to green peach aphid (Myzus persicae).¹⁷ Aphid-derived elicitors mediate ROS burst, which is correlated with Arabidopsis resistance to this phloem sap-feeder.¹⁸ We have recently demonstrated that mutation at BOTRYTIS-INDUCED KINASE1 (BIK1) gene, encoding a receptor-like cytoplasmic kinase in Arabidopsis,^19,20 leads to heightened resistance to aphids.²¹ Aphids feeding on bik1 plants showed less preference, lower population growth and reduced body weight than those on wild-type (WT) Col-0 plants. ROS production and HR-like lesions were detected much earlier and more intensely on aphid-infested bik1 than WT plants. Further investigation revealed that resistance was due to loss of suppression of PAD4, a senescence-promoting gene, in bik1.^17,21

Rapid H₂O₂ production upon aphid infestation in the bik1 mutant suggests that BIK1 affects ROS homeostasis. RESPIRATORY BURST OXIDASE HOMOLOGUE (AtRBOH) genes in Arabidopsis encode NADPH oxidases involved in ROS production in response to infection of bacterial and fungal pathogens, particularly AtRBOHD and AtRBOHF.^10,22 Arabidopsis serine/threonine kinase OXIDATIVE SIGNAL-INDUCIBLE1 (AtOXI1) and zinc finger protein AtZat12 are both marker genes for ROS signaling.^23,24 Arabidopsis CATALASE1 (AtCAT1) and CATALASE2 (AtCAT2) can detoxify H₂O₂ and are induced by abiotic stresses.²⁵ Cytosolic ASCORBATE PEROXIDASE1 (AtAPX1) can also scavenge H₂O₂.²⁵ To begin to understand their possible roles in bik1-mediated resistance to aphids, we performed RT-qPCR analysis to determine expression of these genes related to ROS production, signaling or scavenging (Fig. 1). Aphid infestation induced AtRBOHD and AtRBOHF in both WT and bik1 plants. However, elevated basal transcript levels of both genes were detected in bik1 (Fig. 1A). This higher basal expression could contribute to the faster and stronger ROS accumulation upon aphid feeding.²¹ Similarly, we observed heightened basal expression of the ROS-responsive genes AtOXI1 and AtZat12 in bik1 (Fig. 1B). Their transcriptional activation by aphid treatment suggested that the ROS-responsive pathway(s) is intact in the bik1 mutant. In contrast, no significant differences between WT and bik1 were observed in ROS-scavenging genes (Fig. 1C); while AtAPX1 was upregulated in both genotypes, AtCAT1 and AtCAT2 were repressed. Enhanced ROS production coupled with unchanged detoxification may have contributed to the heightened resistance in bik1, highlighting the importance of ROS in plant defense against aphids. On the other hand, excessive expression of ROS-related genes and presumably other genes normally repressed by BIK1 could be metabolically costly. As a result, bik1 exhibits stunted growth and decreased fertility.^20,21

Figure 1.

Elevated basal expression of ROS-generating (A) and ROS-responsive (B) genes in bik1, but no differential gene expression for ROS-detoxification genes (C). Three-week-old plants were infested with 30 aphids for 0, 3 and 24 h. Total RNAs were extracted from Arabidopsis samples after removal of aphids, followed by RT-qPCR analyses. Gene expression levels at each time point were compared to the levels of the corresponding untreated WT samples, which were arbitrarily set at 1. Transcript fold change is shown as LOG2-transformed bar graphs (means ± SE). Data were analyzed by one-way ANOVA. Duncan's multiple range test was used for pairwise comparisons. Means with different letters are statistically significant (P < 0.05). Each experiment was repeated at least 3 times. AtRBOHD and AtRBOHF, RESPIRATORY BURST OXIDASE HOMOLOGUE D and F; AtOXI1, OXIDATIVE SIGNAL-INDUCIBLE1; AtZAT12; AtCAT1 and AtCAT2, CATALASE1 and 2; AtAPX1, ASCORBATE PEROXIDASE1.

Besides functioning as a signaling molecule in host plants to mediate defense gene activation, ROS may cause direct damage to insect tissues and cells.^9,26,27 Aphids perform poorly on bik1 plants,²¹ but little is known of whether they activate mechanisms to combat increased ROS in ingested plant materials. Comparisons of catalytic activities of antioxidant enzymes in aphids fed on WT and bik1 will shed some light on how aphids respond to oxidative stress. Catalases (CATs) convert H₂O₂ into water and oxygen,²⁸ and glutathione S-transferases (GSTs) detoxify secondary oxidation products generated from ROS reacting with intracellular macromolecules.^29,30 Activity levels of these enzymes in insects are believed to be crucial factors in determining their resistance to a broad spectrum of toxic chemicals.³¹ In the current research, we quantified activities of CAT and GST by spectrophotometric-based enzymatic assays.^32,33 Compared to WT, aphids feeding on the bik1 plants had significantly higher activities for both enzymes examined (Fig. 2). Results indicate that, as in many other insect systems, aphids may also overproduce detoxification enzymes to mitigate oxidative stress. Their ability to cope with such a challenge is most likely accompanied by a fitness cost, consistent with the retarded aphid growth and reproduction.

Figure 2.

Detoxification enzyme activity in green peach aphids feeding on the WT or bik1 plants. Age-synchronized aphid neonates were reared on 4-week-old plants for 7 d. Twenty adult aphids were collected and homogenized in 0.2 M sodium phosphate buffer (pH 7.5). The supernatant was used for enzymatic assays. (A) Catalase (CT) activity was analyzed by rate of H₂O₂ consumption at 240 nm.³³ (B) Glutathione S-transferase (GST) activity was measured by using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate at 340 nm.³² All of the enzyme activities (means ± SE) were normalized by total protein determined by Bradford assay. Experiments were repeated at least 3 times. Data were analyzed by independent t-test. The asterisks indicate significant differences between samples ***(P < 0.001).

Taken together, we have revealed heightened basal expression of ROS-generating and -responsive genes in the bik1 mutant that is hypersensitive to aphid infestation. Furthermore, in response to dietary challenge, aphids increased detoxification capacity, a common mechanism to overcome oxidative stress.³⁴ Plant defense and insect counter-defense strategies apparently take a visible toll on growth and development of both parties under challenge, yet such a trade-off is essential for their survival.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Dr. Tesfaye Mengiste (Purdue University) and Dr. Libo Shan (Texas A&M University) for kindly sharing the mutant plant seeds. We appreciate thoughtful discussions with Dr. Hisashi Koiwa (Department of Horticulture, Texas A&M). We would like to thank Dr. Zivko Nikolov at the Institute for Plant Genomics & Biotechnology, Texas A&M University, for use of the BioTek Microplate Readers.