Sivakumar Swaminathan

CYP76M7 Is an ent-Cassadiene C11α-Hydroxylase Defining a Second Multifunctional Diterpenoid Biosynthetic Gene Cluster in Rice

Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways.

Characterization of CYP76M5–8 Indicates Metabolic Plasticity within a Plant Biosynthetic Gene Cluster

Background: Biosynthetic gene clusters are unusual in plants, yet may provide insight into the associated evolution of secondary metabolism. Results: The cytochromes P450 CYP76M5–8, found in one such cluster, have multiple functions in rice diterpenoid metabolism. Conclusion: Plant biosynthetic gene clusters can encode metabolic plasticity. Significance: Such plasticity may enable further evolution, and be a broader feature of plant secondary metabolism. Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5–8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.

The Fusarium virguliforme Toxin FvTox1 Causes Foliar Sudden Death Syndrome-Like Symptoms in Soybean

Fusarium virguliforme causes sudden death syndrome (SDS) in soybean. The pathogen has never been isolated from diseased foliar tissues; therefore, one or more toxins have been considered to cause foliar SDS development. Cell-free F. virguliforme culture filtrates containing a toxin causes foliar SDS in soybean. A low-molecular-weight protein of approximately 13.5 kDa (FvTox1), purified from F. virguliforme culture filtrates, produces foliar SDS-like symptoms in cut soybean seedlings. Anti-FvTox1 monoclonal antibodies raised against the purified FvTox1 were used in isolating the FvTox1 gene. In the presence of light, recombinant FvTox1 protein expressed in an insect cell line resulted in chlorosis and necrosis in soybean leaf disks that are typical foliar SDS symptoms. SDS-susceptible but not the SDS-resistant soybean lines were sensitive to the baculovirus-expressed toxin. The requirement of light for foliar SDS-like symptom development indicates that FvTox1 induces foliar SDS in soybean, most likely through production of free radicals by interrupting photosynthesis.

Investigation of the Fusarium virguliforme fvtox1 mutants revealed that the FvTox1 toxin is involved in foliar sudden death syndrome development in soybean

The soil borne fungus, Fusariumvirguliforme, causes sudden death syndrome (SDS) in soybean, which is a serious foliar and root rot disease. The pathogen has never been isolated from the diseased foliar tissues; phytotoxins produced by the pathogen are believed to cause foliar SDS symptoms. One of these toxins, a 13.5-kDa acidic protein named FvTox1, has been hypothesized to interfere with photosynthesis in infected soybean plants and cause foliar SDS. The objective of this study is to determine if FvTox1 is involved in foliar SDS development. We created and studied five independent knockout fvtox1 mutants to study the function of FvTox1. We conducted Agrobacteriumtumefaciens-mediated transformation to accomplish homologous recombination of FvTox1 with a hygromycin B resistance gene, hph, to generate the fvtox1 mutants. Approximately 40 hygromycin-resistant transformants were obtained from 106 conidial spores of the F. virguliforme Mont-1 isolate when the spores were co-cultivated with the A. tumefaciens EHA105 but not with LBA4044 strain carrying a recombinant binary plasmid, in which the hph gene encoding hygromycin resistance was flanked by 5′- and 3′-end FvTox1 sequences. We observed homologous recombination-mediated integration of hph into the FvTox1 locus among five independent fvtox1 mutants. In stem-cutting assays using cut soybean seedlings fed with cell-free F. virguliforme culture filtrates, the knockout fvtox1 mutants caused chlorophyll losses and foliar SDS symptoms, which were over twofold less than those caused by the virulent F. virguliforme Mont-1 isolate. Similarly, in root inoculation assays, more than a twofold reduction in foliar SDS development and chlorophyll losses was observed among the seedlings infected with the fvtox1 mutants as compared to the seedlings infected with the wild-type Mont-1 isolate. These results suggest that FvTox1 is a major virulence factor involved in foliar SDS development in soybean. It is expected that interference of the function of this toxin in transgenic soybean plants will lead to generation of SDS-resistant soybean cultivars.

MetNetGE: Visualizing biological networks in hierarchical views and 3D tiered layouts

Linking experimental data with large-scale biomedical networks is key for achieving new discoveries in system biology research. Visualization tools that facilitate these tasks often result in a dense web of connections that resembles a tangled hairball and is difficult to interpret. MetNetGE is an interactive pathway navigation tool based on Google Earth that features novel visualization techniques for pathway information display. Instead of simply showing all the pathways in a network in a complex graph, MetNetGE visualizes the entire network of pathways based on the hierarchical pathway ontology using a novel radial space filling (RSF) method. Orbits show when pathways belong to multiple categories. Mapping cumulative experiment statistics on the RSF drawing aids biologists in easily identifying highly activated pathways in an experiment. After identifying key pathways, biologists can fly to the corresponding region and see the detailed pathway and experimental data in an aligned 3D tiered layout with simplified cross-layer connection patterns.

The plant immunity inducer pipecolic acid accumulates in the xylem sap and leaves of soybean seedlings following Fusarium virguliforme infection.

The causal agent of the soybean sudden death syndrome (SDS), Fusarium virguliforme, remains in infected roots and secretes toxins to cause foliar SDS. In this study we investigated the xylem sap, roots, and leaves of F. virguliforme-infected and -uninfected soybean seedlings for any changes in a set of over 3,000 metabolites following pathogen infection by conducting GC/MS and LC/MS/MS, and detected 273 biochemicals. Levels of many intermediates of the TCA cycle were reduced suggesting suppression of this metabolic pathway by the pathogen. There was an increased accumulation of peroxidated lipids in leaves of F. virguliforme-infected plants suggesting possible involvement of free radicals and lipoxygenases in foliar SDS development. Levels of both isoflavone conjugates and isoflavonoid phytoalexins were decreased in infected roots suggesting degradation of these metabolites by the pathogen to promote root necrosis. The levels of the plant immunity inducer pipecolic acid (Pip) and the plant hormone salicylic acid (SA) were significantly increased in xylem sap (in case of Pip) and leaves (in case of both Pip and SA) of F. virguliforme-infected soybean plants compared to the control plants. This suggests a major signaling role of Pip in inducing host defense responses in above ground parts of the F. virguliforme-infected soybean. Increased accumulation of pipecolic acid in foliar tissues was associated with the induction of GmALD1, the soybean homolog of Arabidopsis ALD1. This metabolomics study generated several novel hypotheses for studying the mechanisms of SDS development in soybean.

‘ MN1606SP’ by ‘Spencer’ filial soybean population reveals novel quantitative trait loci and interactions among loci conditioning SDS resistance

Key messageFour novel QTL and interactions among QTL were identified in this research, using as a parent line the most SDS-resistant genotype within soybean cultivars of the US early maturity groups.AbstractSoybean sudden death syndrome (SDS) reduces soybean yield in most of the growing areas of the world. The causal agent of SDS, soilborne fungus Fusarium virguliforme (Fv), releases phytotoxins taken up by the plant to produce chlorosis and necrosis in the leaves. Planting resistant cultivars is the most successful management practice to control the disease. The objective of this study was to identify quantitative trait loci (QTL) associated with the resistance response of MN1606SP to SDS. A mapping population of F2:3 lines created by crossing the highly resistant cultivar ‘MN1606SP’ and the susceptible cultivar ‘Spencer’ was phenotyped in the greenhouse at three different planting times, each with three replications. Plants were artificially inoculated using SDS infested sorghum homogeneously mixed with the soil. Data were collected on three disease criteria, foliar disease incidence (DI), foliar leaf scorch disease severity (DS), and root rot severity. Disease index (DX) was calculated as DI × DS. Ten QTL were identified for the different disease assessment criteria, three for DI, four for DX, and three for root rot severity. Three QTL identified for root rot severity and one QTL for disease incidence are considered novel, since no previous reports related to these QTL are available. Among QTL, two interactions were detected between four different QTL. The interactions suggest that resistance to SDS is not only dependent on additive gene effects. The novel QTL and the interactions observed in this study will be useful to soybean breeders for improvement of SDS resistance in soybean germplasm.

Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean

Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed.

Arabidopsis Novel Glycine-Rich Plasma Membrane PSS1 Protein Enhances Disease Resistance in Transgenic Soybean Plants

Arabidopsis nonhost resistance gene PSS1 encoding an unknown glycine-rich plasma membrane protein has shown to enhance sudden death syndrome resistance in transgenic soybean plants. Nonhost resistance is defined as the immunity of a plant species to all nonadapted pathogen species. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 is nonhost to the oomycete plant pathogen Phytophthora sojae and the fungal plant pathogen Fusarium virguliforme that are pathogenic to soybean (Glycine max). Previously, we reported generating the pss1 mutation in the pen1-1 genetic background as well as genetic mapping and characterization of the Arabidopsis nonhost resistance Phytophthora sojae-susceptible gene locus, PSS1. In this study, we identified six candidate PSS1 genes by comparing single-nucleotide polymorphisms of (1) the bulked DNA sample of seven F2:3 families homozygous for the pss1 allele and (2) the pen1-1 mutant with Columbia-0. Analyses of T-DNA insertion mutants for each of these candidate PSS1 genes identified the At3g59640 gene encoding a glycine-rich protein as the putative PSS1 gene. Later, complementation analysis confirmed the identity of At3g59640 as the PSS1 gene. PSS1 is induced following P. sojae infection as well as expressed in an organ-specific manner. Coexpression analysis of the available transcriptomic data followed by reverse transcriptase-polymerase chain reaction suggested that PSS1 is coregulated with ATG8a (At4g21980), a core gene in autophagy. PSS1 contains a predicted single membrane-spanning domain. Subcellular localization study indicated that it is an integral plasma membrane protein. Sequence analysis suggested that soybean is unlikely to contain a PSS1-like defense function. Following the introduction of PSS1 into the soybean cultivar Williams 82, the transgenic plants exhibited enhanced resistance to F. virguliforme, the pathogen that causes sudden death syndrome.

Tanscriptomic Study of the Soybean-Fusarium virguliforme Interaction Revealed a Novel Ankyrin-Repeat Containing Defense Gene, Expression of Whose during Infection Led to Enhanced Resistance to the Fungal Pathogen in Transgenic Soybean Plants

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.