Benzoxazinoids in root exudates of maize attract Pseudomonas putida to the rhizosphere.
ABSTRACT: Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.
Project description:The rhizobiome is an important regulator of plant growth and health. Plants shape their rhizobiome communities through production and release of primary and secondary root metabolites. Benzoxazinoids (BXs) are common tryptophan-derived secondary metabolites in grasses that regulate belowground and aboveground biotic interactions. In addition to their biocidal activity, BXs can regulate plant-biotic interactions as semiochemicals or within-plant defence signals. However, the full extent and mechanisms by which BXs shape the root-associated microbiome has remained largely unexplored. Here, we have taken a global approach to examine the regulatory activity of BXs on the maize root metabolome and associated bacterial and fungal communities. Using untargeted mass spectrometry analysis in combination with prokaryotic and fungal amplicon sequencing, we compared the impacts of three genetic mutations in different steps in the BX pathway. We show that BXs regulate global root metabolism and concurrently influence the rhizobiome in a root type-dependent manner. Correlation analysis between BX-controlled root metabolites and bacterial taxa suggested a dominant role for BX-dependent metabolites, particularly flavonoids, in constraining a range of soil microbial taxa, while stimulating methylophilic bacteria. Our study supports a multilateral model by which BXs control root-microbe interactions via a global regulatory function in root secondary metabolism.
Project description:BACKGROUND:Plants actively shape their associated microbial communities by synthesizing bio-active substances. Plant secondary metabolites are known for their signaling and plant defense functions, yet little is known about their overall effect on the plant microbiome. In this work, we studied the effects of benzoxazinoids (BXs), a group of secondary metabolites present in maize, on the host-associated microbial structure. Using BX knock-out mutants and their W22 parental lines, we employed 16S and ITS2 rRNA gene amplicon analysis to characterize the maize microbiome at early growth stages. RESULTS:Rhizo-box experiment showed that BXs affected microbial communities not only in roots and shoots, but also in the rhizosphere. Fungal richness in roots was more affected by BXs than root bacterial richness. Maize genotype (BX mutants and their parental lines) as well as plant age explained both fungal and bacterial community structure. Genotypic effect on microbial communities was stronger in roots than in rhizosphere. Diverse, but specific, microbial taxa were affected by BX in both roots and shoots, for instance, many plant pathogens were negatively correlated to BX content. In addition, a co-occurrence analysis of the root microbiome revealed that BXs affected specific groups of the microbiome. CONCLUSIONS:This study provides insights into the role of BXs for microbial community assembly in the rhizosphere and in roots and shoots. Coupling the quantification of BX metabolites with bacterial and fungal communities, we were able to suggest a gatekeeper role of BX by showing its correlation with specific microbial taxa and thus providing insights into effects on specific fungal and bacterial taxa in maize roots and shoots. Root microbial co-occurrence networks revealed that BXs affect specific microbial clusters.
Project description:Plant disease can be effectively suppressed in intercropping systems. Our previous study demonstrated that neighboring maize plants can restrict the spread of soil-borne pathogens of pepper plants by secreting defense compounds into the soil. However, whether maize plant can receive benefits from its neighboring pepper plants in an intercropping system is little attention. We examined the effects of maize roots treated with elicitors from the pepper pathogen Phytophthora capsici and pepper root exudates on the synthesis of 1,4-benzoxazine-3-ones (BXs), the expression of defense-related genes in maize, and their ability to alleviate the severity of southern corn leaf blight (SCLB) caused by Bipolaris maydis. We found that SCLB was significantly reduced after the above treatments. The contents of 1,4-benzoxazine-3-ones (BXs: DIBOA, DIMBOA, and MBOA) and the expression levels of BX synthesis and defense genes in maize roots and shoots were up-regulated. DIMBOA and MBOA effectively inhibited the mycelium growth of Bipolaris maydis at physiological concentrations in maize shoots. Further studies suggested that the defense related pathways or genes in maize roots and shoots were activated by elicitors from the P. capsici or pepper root exudates. In conclusion, maize increased the levels of BXs and defense gene expression both in roots and shoots after being triggered by root exudates and pathogen from neighboring pepper plants, eventually enhancing its resistance.
Project description:Benzoxazinoids (BXs) are secondary metabolites with diverse functions, but are primarily involved in protecting plants, mainly from the family Poaceae, against insects and fungal pathogens. Rye is a cereal crop that is highly resistant to biotic stresses. However, its susceptibility to brown rust caused by Puccinia recondita f. sp. secalis (Prs) is still a major problem affecting its commercial production. Additionally, the genetic and metabolic factors related to this disease remain poorly characterized. In this study, we investigated whether and to what extent the brown rust infection and the inoculation procedure affect the contents of specific BXs (HBOA, GDIBOA, DIBOA, GDIMBOA, DIMBOA, and MBOA) and the expression of genes related to BX (ScBx1-5, ScIgl, and Scglu). We revealed that treatments with water and a urediniospore suspension usually downregulate gene expression levels. Moreover, HBOA and DIBOA contents decreased, whereas the contents of the remaining metabolites increased. Specifically, the MBOA content increased more after the mock treatment than after the Prs treatment, whereas the increase in GDIBOA and GDIMBOA levels was usually due to the Prs infection, especially at two of the most critical time-points, 17 and 24 h post-treatment. Therefore, GDIBOA and GDIMBOA are glucosides that are important components of rye defence responses to brown rust. Furthermore, along with MBOA, they protect rye against the stress associated with the inoculation procedure used in this study.
Project description:Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription.
Project description:Maize (Zea mays L.) is a staple crop worldwide with extensive genetic variations. Various insects attack maize plants causing large yield loss. Here, we investigated the responses of maize B73, a susceptible line, and Mo17, a resistant line, to the aphid Rhopalosiphum padi on metabolite and transcriptome levels. R. padi feeding had no effect on the levels of the defensive metabolites benzoxazinoids (Bxs) in either line, and Mo17 contained substantially greater levels of Bxs than did B73. Profiling of the differentially expressed genes revealed that B73 and Mo17 responded to R. padi infestation specifically, and importantly, these two lines showed large gene expression differences even without R. padi herbivory. Correlation analysis identified four transcription factors (TFs) that might account for the high Bx levels in Mo17. Similarly, genome-wide alternative splicing (AS) analyses indicated that both B73 and Mo17 had temporally specific responses to R. padi infestation, and these two lines also exhibited large differences of AS regulation under normal condition, and 340 genes, including 10 TFs, were constantly differentially spliced. This study provides large-scale resource datasets for further studies on the mechanisms underlying maize-aphid interactions, and highlights the phenotypic divergence in defense against aphids among maize varieties.
Project description:Tailoring defense responses to different attackers is important for plant performance. Plants can use secondary metabolites with dual functions in resistance and defense signaling to mount herbivore-specific responses. To date, the specificity and evolution of this mechanism are unclear. Here, we studied the functional architecture, specificity, and genetic basis of defense regulation by benzoxazinoids in cereals. We document that DIMBOA-Glc induces callose as an aphid resistance factor in wheat. O-methylation of DIMBOA-Glc to HDMBOA-Glc increases plant resistance to caterpillars but reduces callose inducibility and resistance to aphids. DIMBOA-Glc induces callose in wheat and maize, but not in Arabidopsis, while the glucosinolate 4MO-I3M does the opposite. We identify a wheat O-methyltransferase (TaBX10) that is induced by caterpillar feeding and converts DIMBOA-Glc to HDMBOA-Glc in vitro. While the core pathway of benzoxazinoid biosynthesis is conserved between wheat and maize, the wheat genome does not contain close homologs of the maize DIMBOA-Glc O-methyltransferase genes, and TaBx10 is only distantly related. Thus, the functional architecture of herbivore-specific defense regulation is similar in maize and wheat, but the regulating biosynthetic genes likely evolved separately. This study shows how two different cereal species independently achieved herbivore-specific defense activation by regulating secondary metabolite production.
Project description:Transcriptome profiling of Pseudomonas putida KT2440 comparing cells exposed for 1 hour to DIMBOA from maize (Zea mays) to unexposed cells Two-condition experiment, unexposed vs. exposed, three biological replicates independantly grown and harvested. A dye swap was performed on each biological replicate for a total of six hybridizations. Each GEO Sample represents the data after merging of the technical (dye swap) replicates.
Project description:Plant root colonization by rhizobacteria can protect plants against pathogens and promote plant growth, and chemotaxis to root exudates was shown to be an essential prerequisite for efficient root colonization. Since many chemoattractants control the transcript levels of their cognate chemoreceptor genes, we have studied here the transcript levels of the 27 Pseudomonas putida KT2440 chemoreceptor genes in the presence of different maize root exudate (MRE) concentrations. Transcript levels were increased for 10 chemoreceptor genes at low MRE concentrations, whereas almost all receptor genes showed lower transcript levels at high MRE concentrations. The exposure of KT2440 to different MRE concentrations did not alter c-di-GMP levels, indicating that changes in chemoreceptor transcripts are not mediated by this second messenger. Data suggest that rhizosphere colonization unfolds in a temporal fashion. Whereas at a distance to the root, exudates enhance chemoreceptor gene transcript levels promoting in turn chemotaxis, this process is reversed in root vicinity, where the necessity of chemotaxis toward the root may be less important. Insight into KT2440 signaling processes were obtained by analyzing mutants defective in the three cheA paralogous genes. Whereas a mutant in cheA1 showed reduced c-di-GMP levels and impaired biofilm formation, a cheA2 mutant was entirely deficient in MRE chemotaxis, indicating the existence of homologs of the P. aeruginosa wsp and che (chemotaxis) pathways. Signaling through both pathways was important for efficient maize root colonization. Future studies will show whether the MRE concentration dependent effect on chemoreceptor gene transcript levels is a feature shared by other species.
Project description:The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye.