Legumes Regulate Symbiosis with Rhizobia via Their Innate Immune System.

Plant roots are constantly exposed to a diverse microbiota of pathogens and mutualistic partners. The host's immune system is an essential component for its survival, enabling it to monitor nearby microbes for potential threats and respond with a defence response when required. Current research suggests that the plant immune system has also been employed in the legume-rhizobia symbiosis as a means of monitoring different rhizobia strains and that successful rhizobia have evolved to overcome this system to infect the roots and initiate nodulation. With clear implications for host-specificity, the immune system has the potential to be an important target for engineering versatile crops for effective nodulation in the field. However, current knowledge of the interacting components governing this pathway is limited, and further research is required to build on what is currently known to improve our understanding. This review provides a general overview of the plant immune system's role in nodulation. With a focus on the cycles of microbe-associated molecular pattern-triggered immunity (MTI) and effector-triggered immunity (ETI), we highlight key molecular players and recent findings while addressing the current knowledge gaps in this area.

Spatiotemporal changes in gibberellin content are required for soybean nodulation.

The plant hormone gibberellin (GA) is required at different stages of legume nodule development, with its spatiotemporal distribution tightly regulated. Transcriptomic and bioinformatic analyses established that several key GA biosynthesis and catabolism enzyme encoding genes are critical to soybean (Glycine max) nodule formation. We examined the expression of several GA oxidase genes and used a Förster resonance energy transfer-based GA biosensor to determine the bioactive GA content of roots inoculated with DsRed-labelled Bradyrhizobium diazoefficiens. We manipulated the level of GA by genetically disrupting the expression of GA oxidase genes. Moreover, exogenous treatment of soybean roots with GA3 induced the expression of key nodulation genes and altered infection thread and nodule phenotypes. GmGA20ox1a, GmGA3ox1a, and GmGA2ox1a are upregulated in soybean roots inoculated with compatible B. diazoefficiens. GmGA20ox1a expression is predominately localized to the transient meristem of soybean nodules and coincides with the spatiotemporal distribution of bioactive GA occurring throughout nodule organogenesis. GmGA2ox1a exhibits a nodule vasculature-specific expression pattern, whereas GmGA3ox1a can be detected throughout the nodule and root. Disruptions in the level of GA resulted in aberrant rhizobia infection and reduced nodule numbers. Collectively, our results establish a central role for GAs in root hair infection by symbiotic rhizobia and in nodule organogenesis.

Characterisation of Medicago truncatula CLE34 and CLE35 in nitrate and rhizobia regulation of nodulation.

Legumes form a symbiosis with atmospheric nitrogen (N2 )-fixing soil rhizobia, resulting in new root organs called nodules that enable N2 -fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been investigated in Medicago truncatula, for which only rhizobia-induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. MtCLE35 was found to inhibit nodulation in a SUNN- and RDN1-dependent manner via overexpression analysis. Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.

Shoot-derived miR2111 controls legume root and nodule development.

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture.

A differential k-mer analysis pipeline for comparing RNA-Seq transcriptome and meta-transcriptome datasets without a reference.

Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

Legume nodulation: The host controls the party.

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen-fixing rhizobia bacteria is central to their advantage. This plant-microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.

Triarabinosylation is required for nodulation-suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum.

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.

Hypernodulating soybean mutant line nod4 lacking 'Autoregulation of Nodulation' (AON) has limited root-to-shoot water transport capacity.

BACKGROUND AND AIMS: Although hypernodulating phenotype mutants of legumes, such as soybean, possess a high leaf N content, the large number of root nodules decreases carbohydrate availability for plant growth and seed yield. In addition, under conditions of high air vapour pressure deficit (VPD), hypernodulating plants show a limited capacity to replace water losses through transpiration, resulting in stomatal closure, and therefore decreased net photosynthetic rates. Here, we used hypernodulating (nod4) (282.33 ± 28.56 nodules per plant) and non-nodulating (nod139) (0 nodules per plant) soybean mutant lines to determine explicitly whether a large number of nodules reduces root hydraulic capacity, resulting in decreased stomatal conductance and net photosynthetic rates under high air VPD conditions. METHODS: Plants were either inoculated or not inoculated with Bradyrhizobium diazoefficiens (strain BR 85, SEMIA 5080) to induce nitrogen-fixing root nodules (where possible). Absolute root conductance and root conductivity, plant growth, leaf water potential, gas exchange, chlorophyll a fluorescence, leaf 'greenness' [Soil Plant Analysis Development (SPAD) reading] and nitrogen content were measured 37 days after sowing. KEY RESULTS: Besides the reduced growth of hypernodulating soybean mutant nod4, such plants showed decreased root capacity to supply leaf water demand as a consequence of their reduced root dry mass and root volume, which resulted in limited absolute root conductance and root conductivity normalized by leaf area. Thereby, reduced leaf water potential at 1300 h was observed, which contributed to depression of photosynthesis at midday associated with both stomatal and non-stomatal limitations. CONCLUSIONS: Hypernodulated plants were more vulnerable to VPD increases due to their limited root-to-shoot water transport capacity. However, greater CO2 uptake caused by the high N content can be partly compensated by the stomatal limitation imposed by increased VPD conditions.

Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation.

MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean.

Molecular Signals in Nodulation Control.

Our world is facing major problems relating to food production. According to an August 30, 2015 program of LANDLINE (Australian Broadcasting Corporation, Australia),we lose 120,000,000 hectares of agricultural land per year due to population growth, associated urbanisation, and desertification.

MicroRNA167-Directed Regulation of the Auxin Response Factors GmARF8a and GmARF8b Is Required for Soybean Nodulation and Lateral Root Development.

Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes nodule inception, nodulation signaling pathway1, early nodulin40-1, NF-YA1 (previously known as HAEM activator protein2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean.

Identification of the primary lesion of toxic aluminum in plant roots.

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.

Genome-wide annotation and characterization of CLAVATA/ESR (CLE) peptide hormones of soybean (Glycine max) and common bean (Phaseolus vulgaris), and their orthologues of Arabidopsis thaliana.

CLE peptides are key regulators of cell proliferation and differentiation in plant shoots, roots, vasculature, and legume nodules. They are C-terminally encoded peptides that are post-translationally cleaved and modified from their corresponding pre-propeptides to produce a final ligand that is 12-13 amino acids in length. In this study, an array of bionformatic and comparative genomic approaches was used to identify and characterize the complete family of CLE peptide-encoding genes in two of the world's most important crop species, soybean and common bean. In total, there are 84 CLE peptide-encoding genes in soybean (considerably more than the 32 present in Arabidopsis), including three pseudogenes and two multi-CLE domain genes having six putative CLE domains each. In addition, 44 CLE peptide-encoding genes were identified in common bean. In silico characterization was used to establish all soybean homeologous pairs, and to identify corresponding gene orthologues present in common bean and Arabidopsis. The soybean CLE pre-propeptide family was further analysed and separated into seven distinct groups based on structure, with groupings strongly associated with the CLE domain sequence and function. These groups provide evolutionary insight into the CLE peptide families of soybean, common bean, and Arabidopsis, and represent a novel tool that can aid in the functional characterization of the peptides. Transcriptional evidence was also used to provide further insight into the location and function of all CLE peptide-encoding members currently available in gene atlases for the three species. Taken together, this in-depth analysis helped to identify and categorize the complete CLE peptide families of soybean and common bean, established gene orthologues within the two legume species, and Arabidopsis, and provided a platform to help compare, contrast, and identify the function of critical CLE peptide hormones in plant development.

The soybean (Glycine max) nodulation-suppressive CLE peptide, GmRIC1, functions interspecifically in common white bean (Phaseolus vulgaris), but not in a supernodulating line mutated in the receptor PvNARK.

Legume plants regulate the number of nitrogen-fixing root nodules they form via a process called the Autoregulation of Nodulation (AON). Despite being one of the most economically important and abundantly consumed legumes, little is known about the AON pathway of common bean (Phaseolus vulgaris). We used comparative- and functional-genomic approaches to identify central components in the AON pathway of common bean. This includes identifying PvNARK, which encodes a LRR receptor kinase that acts to regulate root nodule numbers. A novel, truncated version of the gene was identified directly upstream of PvNARK, similar to Medicago truncatula, but not seen in Lotus japonicus or soybean. Two mutant alleles of PvNARK were identified that cause a classic shoot-controlled and nitrate-tolerant supernodulation phenotype. Homeologous over-expression of the nodulation-suppressive CLE peptide-encoding soybean gene, GmRIC1, abolished nodulation in wild-type bean, but had no discernible effect on PvNARK-mutant plants. This demonstrates that soybean GmRIC1 can function interspecifically in bean, acting in a PvNARK-dependent manner. Identification of bean PvRIC1, PvRIC2 and PvNIC1, orthologues of the soybean nodulation-suppressive CLE peptides, revealed a high degree of conservation, particularly in the CLE domain. Overall, our work identified four new components of bean nodulation control and a truncated copy of PvNARK, discovered the mutation responsible for two supernodulating bean mutants and demonstrated that soybean GmRIC1 can function in the AON pathway of bean.

Rhizobial and mycorrhizal symbioses in Lotus japonicus require lectin nucleotide phosphohydrolase, which acts upstream of calcium signaling.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

The role of symbiotic nitrogen fixation in sustainable production of biofuels.

With the ever-increasing population of the world (expected to reach 9.6 billion by 2050), and altered life style, comes an increased demand for food, fuel and fiber. However, scarcity of land, water and energy accompanied by climate change means that to produce enough to meet the demands is getting increasingly challenging. Today we must use every avenue from science and technology available to address these challenges. The natural process of symbiotic nitrogen fixation, whereby plants such as legumes fix atmospheric nitrogen gas to ammonia, usable by plants can have a substantial impact as it is found in nature, has low environmental and economic costs and is broadly established. Here we look at the importance of symbiotic nitrogen fixation in the production of biofuel feedstocks; how this process can address major challenges, how improving nitrogen fixation is essential, and what we can do about it.

Mechanistic action of gibberellins in legume nodulation.

Legume plants are capable of entering into a symbiotic relationship with rhizobia bacteria. This results in the formation of novel organs on their roots, called nodules, in which the bacteria capture atmospheric nitrogen and provide it as ammonium to the host plant. Complex molecular and physiological changes are involved in the formation and establishment of such nodules. Several phytohormones are known to play key roles in this process. Gibberellins (gibberellic acids; GAs), a class of phytohormones known to be involved in a wide range of biological processes (i.e., cell elongation, germination) are reported to be involved in the formation and maturation of legume nodules, highlighted by recent transcriptional analyses of early soybean symbiotic steps. Here, we summarize what is currently known about GAs in legume nodulation and propose a model of GA action during nodule development. Results from a wide range of studies, including GA application, mutant phenotyping, and gene expression studies, indicate that GAs are required at different stages, with an optimum, tightly regulated level being key to achieve successful nodulation. Gibberellic acids appear to be required at two distinct stages of nodulation: (i) early stages of rhizobia infection and nodule primordium establishment; and (ii) later stages of nodule maturation.

Systemic regulation of soybean nodulation by acidic growth conditions.

Mechanisms inhibiting legume nodulation by low soil pH, although highly prevalent and economically significant, are poorly understood. We addressed this in soybean (Glycine max) using a combination of physiological and genetic approaches. Split-root and grafting studies using an autoregulation-of-nodulation-deficient mutant line, altered in the autoregulation-of-nodulation receptor kinase GmNARK, determined that a systemic, shoot-controlled, and GmNARK-dependent mechanism was critical for facilitating the inhibitory effect. Acid inhibition was independent of aluminum ion concentration and occurred early in nodule development, between 12 and 96 h post inoculation with Bradyrhizobium japonicum. Biological effects were confirmed by measuring transcript numbers of known early nodulation genes. Transcripts decreased on both sides of split-root systems, where only one side was subjected to low-pH conditions. Our findings enhance the present understanding of the innate mechanisms regulating legume nodulation control under acidic conditions, which could benefit future attempts in agriculture to improve nodule development and biological nitrogen fixation in acid-stressed soils.

Structure-function analysis of the GmRIC1 signal peptide and CLE domain required for nodulation control in soybean.

Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela.

KEY MESSAGE: Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.