Genetic Variation, DIMBOA Accumulation, and Candidate Gene Identification in Maize Multiple Insect-Resistance.

Maize seedlings contain high amounts of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and the effect of DIMBOA is directly associated with multiple insect-resistance against insect pests such as Asian corn borer and corn leaf aphids. Although numerous genetic loci for multiple insect-resistant traits have been identified, little is known about genetic controls regarding DIMBOA content. In this study, the best linear unbiased prediction (BLUP) values of DIMBOA content in two ecological environments across 310 maize inbred lines were calculated; and their phenotypic data and BLUP values were used for marker-trait association analysis. We identified nine SSRs that were significantly associated with DIMBOA content, which explained 4.30-20.04% of the phenotypic variation. Combined with 47 original genetic loci from previous studies, we detected 19 hot loci and approximately 11 hot loci (in Bin 1.04, Bin 2.00-2.01, Bin 2.03-2.04, Bin 4.00-4.03, Bin 5.03, Bin 5.05-5.07, Bin 8.01-8.03, Bin 8.04-8.05, Bin 8.06, Bin 9.01, and Bin 10.04 regions) supported pleiotropy for their association with two or more insect-resistant traits. Within the 19 hot loci, we identified 49 candidate genes, including 12 controlling DIMBOA biosynthesis, 6 involved in sugar metabolism/homeostasis, 2 regulating peroxidases activity, 21 associated with growth and development [(auxin-upregulated RNAs (SAUR) family member and v-myb avian myeloblastosis viral oncogene homolog (MYB)], and 7 involved in several key enzyme activities (lipoxygenase, cysteine protease, restriction endonuclease, and ubiquitin-conjugating enzyme). The synergy and antagonism interactions among these genes formed the complex defense mechanisms induced by multiple insect pests. Moreover, sufficient genetic variation was reported for DIMBOA performance and SSR markers in the 310 tested maize inbred lines, and 3 highly (DIMBOA content was 402.74-528.88 µg g-1 FW) and 15 moderate (DIMBOA content was 312.92-426.56 µg g-1 FW) insect-resistant genotypes were major enriched in the Reid group. These insect-resistant inbred lines can be used as parents in maize breeding programs to develop new varieties.

Host plant adaptability and proteomic differences of diverse Rhopalosiphum maidis (Fitch) lineages.

Corn leaf aphid Rhopalosiphum maidis (Fitch) can feed on various cereal crops and transmit viruses that may cause serious economic losses. To test the impact of both host plant species and age on R. maidis, as well as the proteomic difference of diverse populations, we first investigated the survival and reproduction of six R. maidis populations (i.e., LF, HF, GZ, DY, BJ, and MS) via a direct observation method in the laboratory on 10 and 50 cm high maize seedlings, and 10 cm high barley seedlings. Then a proteomic approach was implemented to identify the differentially expressed proteins from both aphids and endosymbionts of BJ and MS populations. Results indicated that the BJ population performed significantly better than the others on both barley and 50 cm high maize seedlings, while no population could survive on 10 cm high maize seedlings. The proteomic results demonstrated that the expression levels of myosin heavy chain (muscle isoform X12) (spot 781) and peroxidase (spot 1383) were upregulated, while ATP-dependent protease Hsp 100 (spot 2137) from Hamiltonella defensa and protein SYMBAF (spot 2703) from Serratia symbiotica were downregulated in the BJ population when compared to expression levels of the MS population. We hypothesize that the fatalness observed on 10 cm high maize seedlings may be caused by secondary metabolites that are synthesized by the seedlings and the MS population of R. maidis should be more stress-resistant than the BJ population. Our results also provide insights for understanding the interaction between host plants and aphids.

Transcriptomic and volatile signatures associated with maize defense against corn leaf aphid.

BACKGROUND: Maize (Zea mays L.) is a major cereal crop, with the United States accounting for over 40% of the worldwide production. Corn leaf aphid [CLA; Rhopalosiphum maidis (Fitch)] is an economically important pest of maize and several other monocot crops. In addition to feeding damage, CLA acts as a vector for viruses that cause devastating diseases in maize. We have shown previously that the maize inbred line Mp708, which was developed by classical plant breeding, provides heightened resistance to CLA. However, the transcriptomic variation conferring CLA resistance to Mp708 has not been investigated. RESULTS: In this study, we contrasted the defense responses of the resistant Mp708 genotype to those of the susceptible Tx601 genotype at the transcriptomic (mRNA-seq) and volatile blend levels. Our results suggest that there was a greater transcriptomic remodeling in Mp708 plants in response to CLA infestation compared to the Tx601 plants. These transcriptomic signatures indicated an activation of hormonal pathways, and regulation of sesquiterpenes and terpenoid synthases in a constitutive and inducible manner. Transcriptomic analysis also revealed that the resistant Mp708 genotype possessed distinct regulation of ethylene and jasmonic acid pathways before and after aphid infestation. Finally, our results also highlight the significance of constitutive production of volatile organic compounds (VOCs) in Mp708 and Tx601 plants that may contribute to maize direct and/or indirect defense responses. CONCLUSIONS: This study provided further insights to understand the role of defense signaling networks in Mp708's resistance to CLA.

Asymmetry in Herbivore Effector Responses: Caterpillar Frass Effectors Reduce Performance of a Subsequent Herbivore.

Multiple species of phytophagous insects may co-occur on a plant and while plants can defend themselves from insect herbivory, plant responses to damage by different species and feeding guilds of insects may be asymmetric. Plants can trigger specific responses to elicitors/effectors in insect secretions altering herbivore performance. Recently, maize chitinases present in fall armyworm (FAW, Spodoptera frugiperda) frass were shown to act as effectors suppressing caterpillar-induced defenses in maize while increasing caterpillar performance. We investigated the effect of frass chitinase-mediated suppression of herbivore defenses in maize on the performance and preference of a subsequent insect herbivore from a different feeding guild, corn leaf aphid (Rhopalosiphum maidis). Aphid performance was highest on plants with FAW damage without frass chitinases compared to damaged plants with frass chitinases or undamaged plants. Plant exposure to frass chitinases post FAW damage also altered the production of herbivore-induced volatile compounds compared to damaged, buffer-treated plants. However, aphid preference to damaged, frass chitinase-treated plants was not different from damaged, buffer-treated plants or undamaged plants. This study suggests that frass effector-mediated alteration of plant defenses affects insect herbivores asymmetrically; while it enhances the performance of caterpillars, it suppresses the performance of subsequent herbivores from a different feeding guild.

Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.

In this study we examined the role of sorghum flavonoids in providing resistance against corn leaf aphid (CLA) Rhopalosiphum maidis. In sorghum, accumulation of these flavonoids is regulated by a MYB transcription factor, yellow seed1 (y1). Functional y1 alleles accumulate 3-deoxyflavonoids (3-DFs) and 3-deoxyanthocyanidins (3-DAs) whereas null y1 alleles fail to accumulate these compounds. We found that significantly higher numbers of alate CLA adults colonized null y1 plants as compared to functional y1 plants. Controlled cage experiments and pairwise choice assays demonstrated that apterous aphids preferred to feed and reproduce on null y1 plants. These near-isogenic sorghum lines do not differ in their epicuticular wax content and were also devoid of any leaf trichomes. Significantly higher mortality of CLA was observed on artificial aphid diet supplemented with flavonoids obtained from functional y1 plants as compared to null y1 plants or the relevant controls. Our results demonstrate that the proximate mechanism underlying the deleterious effects on aphids is y1-regulated flavonoids which are important defense compounds against CLA.

Maize terpene synthase 1 impacts insect behavior via the production of monoterpene volatiles ß-myrcene and linalool.

Plant-derived volatiles are important mediators of plant-insect interactions as they can provide cues for host location and quality, or act as direct or indirect defense molecules. The volatiles produced by Zea mays (maize) include a range of terpenes, likely produced by several of the terpene synthases (TPS) present in maize. Determining the roles of specific terpene volatiles and individual TPSs in maize-insect interactions is challenging due to the promiscuous nature of TPSs in vitro and their potential for functional redundancy. In this study, we used metabolite GWAS of a sweetcorn diversity panel infested with Spodoptera frugiperda (fall armyworm) to identify genetic correlations between TPSs and individual volatiles. This analysis revealed a correlation between maize terpene synthase 1 (ZmTPS1) and emission of the monoterpene volatiles linalool and ß-myrcene. Electroantennogram assays showed gravid S. frugiperda could detect both linalool and ß-myrcene. Quantification of headspace volatiles in a maize tps1 loss-of-function mutant confirmed that ZmTPS1 is an important contributor to linalool and ß-myrcene emission in maize. Furthermore, pairwise choice assays between tps1 mutant and wild-type plants showed that ZmTPS1, and by extension its volatile products, aid host location in the chewing insect S. frugiperda, yet repel the sap-sucking pest, Rhopalosiphum maidis (corn leaf aphid). On the other hand, ZmTPS1 had no impact on indirect defense via the recruitment of the parasitoid Cotesia marginiventris. ZmTPS1 is therefore an important mediator of the interactions between maize and its insect pests.

Evaluation of the Stability of a 1,8-Cineole Nanoemulsion and Its Fumigant Toxicity Effect against the Pests Tetranychus urticae, Rhopalosiphum maidis and Bemisia tabaci.

Pest control is a main concern in agriculture. Indiscriminate application of synthetic pesticides has caused negative impacts leading to the rapid development of resistance in arthropod pests. Plant secondary metabolites have been proposed as a safer alternative to conventional pesticides. Monoterpenoids have reported bioactivities against important pests; however, due to their high volatility, low water solubility and chemical instability, the application of these compounds has been limited. Nanosystems represent a potential vehicle for the broad application of monoterpenoids. In this study, an 1,8-cineole nanoemulsion was prepared by the low energy method of phase inversion, characterization of droplet size distribution and polydispersity index (PDI) was carried out by dynamic light scattering and stability was evaluated by centrifugation and Turbiscan analysis. Fumigant bioactivity was evaluated against Tetranychus urticae, Rhopalosiphum maidis and Bemisia tabaci. A nanoemulsion with oil:surfactant:water ratio of 0.5:1:8.5 had a droplet size of 14.7 nm and PDI of 0.178. Formulation was stable after centrifugation and the Turbiscan analysis showed no particle migration and a delta backscattering of ±1%. Nanoemulsion exhibited around 50% more bioactivity as a fumigant on arthropods when compared to free monoterpenoid. These results suggest that nanoformulations can provide volatile compounds of protection against volatilization, improving their bioactivity.

Inhibition of Rhopalosiphum maidis (Corn Leaf Aphid) Growth on Maize by Virus-Induced Gene Silencing with Sugarcane Mosaic Virus.

The corn leaf aphid (Rhopalosiphum maidis), a damaging pest of maize (Zea mays), is not controlled by the insecticidal proteins in commercially available transgenic crop varieties. One promising approach is to reduce aphid growth and fecundity by targeting the expression of essential genes using plant-mediated RNA interference (RNAi). Here we describe a method whereby Sugarcane Mosaic Virus (SCMV), a positive-strand RNA virus in the Potyviridae family, is used for virus-induced gene silencing (VIGS) of gene expression in R. maidis. A segment of the R. maidis target gene is cloned into SCMV, maize plants are infected with the transgenic virus, aphids are placed on the virus-infected plants and, after a few days of feeding, decreases in target gene expression and aphid reproduction are assessed. This VIGS method can be used for rapid screening of suitable RNAi targets for aphid pest control, as well as to study the in vivo function of specific aphid genes.

Maize defense elicitor, 12-oxo-phytodienoic acid, prolongs aphid salivation.

12-Oxo-phytodienoic acid (OPDA), an intermediate in the jasmonic acid (JA) biosynthesis pathway, regulates diverse signaling functions in plants, including enhanced resistance to insect pests. We previously demonstrated that OPDA promoted enhanced callose accumulation and heightened resistance to corn leaf aphid (CLA; Rhopalosiphum maidis), a phloem sap-sucking insect pest of maize (Zea mays). In this study, we used the electrical penetration graph (EPG) technique to monitor and quantify the different CLA feeding patterns on the maize JA-deficient 12-oxo-phytodienoic acid reductase (opr7opr8) plants. CLA feeding behavior was unaffected on B73, opr7opr8 control plants (- OPDA), and opr7opr8 plants that were pretreated with OPDA (+ OPDA). However, exogenous application of OPDA on opr7opr8 plants prolonged aphid salivation, a hallmark of aphids' ability to suppress the plant defense responses. Collectively, our results indicate that CLA utilizes its salivary secretions to suppress or unplug the OPDA-mediated sieve element occlusions in maize.

Genome sequence of the corn leaf aphid (Rhopalosiphum maidis Fitch).

BACKGROUND: The corn leaf aphid (Rhopalosiphum maidis Fitch) is the most economically damaging aphid pest on maize (Zea mays), one of the world's most important grain crops. In addition to causing direct damage by removing photoassimilates, R. maidis transmits several destructive maize viruses, including maize yellow dwarf virus, barley yellow dwarf virus, sugarcane mosaic virus, and cucumber mosaic virus. FINDINGS: The genome of a parthenogenetically reproducing R. maidis clone was assembled with a combination of Pacific Biosciences (207-fold coverage) and Illumina (83-fold coverage) sequencing. The 689 assembled contigs, which have an N50 size of 9.0 megabases (Mb) and a low level of heterozygosity, were clustered using Phase Genomics Hi-C interaction maps. Consistent with the commonly observed 2n = 8 karyotype of R. maidis, most of the contigs (473 spanning 321 Mb) were successfully oriented into 4 scaffolds. The genome assembly captured the full length of 95.8% of the core eukaryotic genes, indicating that it is highly complete. Repetitive sequences accounted for 21.2% of the assembly, and a total of 17,629 protein-coding genes were predicted with integrated evidence from ab initio and homology-based gene predictions and transcriptome sequences generated with both Pacific Biosciences and Illumina. An analysis of likely horizontally transferred genes identified 2 from bacteria, 7 from fungi, 2 from protozoa, and 9 from algae. Repeat elements, transposons, and genes encoding likely detoxification enzymes (cytochrome P450s, glutathione S-transferases, carboxylesterases, uridine diphosphate-glucosyltransferases, and ABC transporters) were identified in the genome sequence. Other than Buchnera aphidicola (642,929 base pairs, 602 genes), no endosymbiont bacteria were found in R. maidis. CONCLUSIONS: A high-quality R. maidis genome was assembled at the chromosome level. This genome sequence will enable further research related to ecological interactions, virus transmission, pesticide resistance, and other aspects of R. maidis biology. It also serves as a valuable resource for comparative investigation of other aphid species.

Reduction of Plant Suitability for Corn Leaf Aphid (Hemiptera: Aphididae) Under Elevated Carbon Dioxide Condition.

In the current context of global climate change, atmospheric carbon dioxide (CO2) concentrations are continuously rising with potential influence on plant-herbivore interactions. The effect of elevated CO2 (eCO2) on feeding behavior of corn leaf aphid, Rhopalosiphum maidis (Fitch) on barley seedlings Hordeum vulgare L. was tracked using electrical penetration graph (EPG). The nutrient content of host plant and the developmental indexes of aphids under eCO2 and ambient CO2 (aCO2) conditions were also investigated. Barley seedlings under eCO2 concentration had lower contents of crude protein and amino acids. EPG analysis showed the plants cultivated under eCO2 influenced the aphid feeding behavior, by prolonging the total pre-probation time of the aphids (wandering and locating the feeding site) and the ingestion of passive phloem sap. Moreover, fresh body weight, fecundity and intrinsic population growth rate of R. maidis was significantly decreased in eCO2 in contrast to aCO2 condition. Our findings suggested that changes in plant nutrition caused by eCO2, mediated via the herbivore host could affect insect feeding behavior and population dynamics.

Effects of Host Plants Reared under Elevated CO2 Concentrations on the Foraging Behavior of Different Stages of Corn Leaf Aphids Rhopalosiphum maidis.

Climate change is a major environmental concern and is directly related to the increasing concentrations of greenhouse gases. The increase in concentrations of atmospheric carbon dioxide (CO2), not only affects plant growth and development, but also affects the emission of plant organic volatile compounds (VOCs). Changes in the plant odor profile may affect the plant-insect interactions, especially the behavior of herbivorous insects. In this study, we compared the foraging behavior of corn leaf aphid (Rhopalosiphum maidis) on barley (Hordeum vulgare L.) seedlings grown under contrasted CO2 concentrations. During the dual choice bioassays, the winged and wingless aphids were more attracted by the VOCs of barley seedlings cultivated under ambient CO2 concentrations (aCO2; 450 ppm) than barley seedlings cultivated under elevated CO2 concentrations (eCO2; 800 ppm), nymphs were not attracted by the VOCs of eCO2 barley seedlings. Then, volatile compositions from 14-d-old aCO2 and eCO2 barley seedlings were investigated by GC-MS. While 16 VOCs were identified from aCO2 barley seedlings, only 9 VOCs were found from eCO2 barley seedlings. At last, we discussed the potential role of these chemicals observed during choice bioassays. Our findings lay foundation for functional response of corn leaf aphid under climate change through host plant modifications.

Intraplant communication in maize contributes to defense against insects.

The vasculature of plants act as a channel for transport of signal(s) that facilitate long-distance intraplant communication. In maize, Maize insect resistance1-Cysteine Protease (Mir1-CP), which has homology to papain-like proteases, provides defense to different feeding guilds of insect pests. Furthermore, accumulation of Mir1-CP in the vasculature suggests that Mir1-CP can potentially function as a phloem-mobile protein. In a recent study, we provided evidence that Mir1-CP can curtail the growth of phloem-sap sucking insect, corn leaf aphid (CLA; Rhopalosiphum maidis). Our current study further examined whether aboveground feeding by CLA can induce resistance to subsequent herbivory by belowground feeding western corn rootworm (WCR; Diabrotica virgifera virgifera). Aboveground feeding by CLA systemically induced the accumulation of Mir1-CP in the roots. Furthermore, foliage feeding by CLA provided enhanced resistance to subsequent herbivory by belowground feeding of WCR. Taken together, our previous findings and results presented here indicate that long-distance transport of Mir1-CP is critical for providing enhanced resistance to insect attack in maize.